Development of an apoptosis-assisted decellularization method for maximal preservation of nerve tissue structure.

Preservation of tissue structure is often a primary goal when optimizing tissue and organ decellularization methods. Many current protocols nonetheless rely on detergents that aid extraction of cellular components but also damage tissue architecture. It may be more beneficial to leverage an innate cellular process such as apoptosis and promote cell removal without the use of damaging reagents. During apoptosis, a cell detaches from the extracellular matrix, degrades its internal components, and fragments its contents for easier clearance. We have developed a method that leverages this process to achieve tissue decellularization using only mild wash buffers. We have demonstrated that treating peripheral nerve tissue with camptothecin induced both an early marker of apoptosis, cleaved caspase-3 expression, as well as a late stage marker, TUNEL+ DNA fragmentation. Clearance of the cellular components was then achieved in an apoptosis-dependent manner using a gentle wash in hypertonic phosphate buffered saline followed by DNase treatment. This wash paradigm did not significantly affect collagen or glycosaminoglycan content, but it was sufficient to remove any trace of the cytotoxic compound based on conditioned media experiments. The resulting acellular tissue graft was immunogenically tolerated in vivo and exhibited an intact basal lamina microarchitecture mimicking that of native, unprocessed nerve. Hence, ex vivo induction of apoptosis is a promising method to decellularize tissue without the use of harsh reagents while better preserving the benefits of native tissue such as tissue-specific composition and microarchitecture. STATEMENT OF SIGNIFICANCE: Tissue decellularization has expanded the ability to generate non-immunogenic organ replacements for a broad range of health applications. Current technologies typically rely on the use of harsh agents for clearing cellular debris, altering the tissue structure and potentially diminishing the pro-regenerative effects. We have developed a method for effectively, yet gently, removing cellular components from peripheral nerve tissue while preserving the native tissue architecture. The novelty of this process is in the induction of programmed cell death - or apoptosis - via a general cytotoxin, thereby enabling antigen clearance using only hypertonic wash buffers. The resulting acellular nerve scaffolds are nearly identical to unprocessed tissue on a microscopic level and elicit low immune responses comparable to an isograft negative control in a model of subcutaneous implantation.

Improved perfusion after subcritical ischemia in muscle flaps treated with vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF), a potent endothelial mitogen, is secreted in ischemic tissue and plays a pivotal role in angiogenesis. We studied whether VEGF administered to a rat muscle flap at the time of ischemia induction would increase microcirculatory flow to the flap. The cremaster muscle flap was isolated on its neurovascular pedicle. Ischemia was induced by clamping the vascular pedicle, and 0.2 ml of either VEGF (0.1 microg) or vehicle (phosphate-buffered saline) was immediately infused into the muscle. After 4 or 6 hours, the clamps were released, and the cremaster was placed in a pocket in the medial thigh for 24 hours. The muscle was then dissected, and microcirculatory measurements were made under intravital microscopy. Six animals were used in each of the four groups. All flaps exposed to 6 hours of ischemia, the duration considered to be critical ischemia, had no significant microcirculatory flow, regardless of treatment with VEGF. In the 4-hour ischemia group, or subcritical ischemia group, red blood cell velocity in arterioles was 14 mm/sec in muscles treated with VEGF and 9 mm/sec in controls (p = 0.02), and capillary flow was 7 per high-power field in muscles treated with VEGF versus 2 per high-power field in controls (p = 0.0005). Thus, VEGF did not alter microcirculatory flow in a muscle flap exposed to critical ischemia, but it did enhance flow to a flap exposed to subcritical ischemia.

Microcirculatory window for early detection of allograft rejection.

The purpose of this study is to introduce a technical detail on a transplantation model for in vivo evaluation of microcirculatory changes during the acute phase of allograft rejection. The cremaster muscle is incorporated and transplanted along with the hind limb to detect and study ischemia/reperfusion injury and the acute phase of allograft rejection in rats. Thirty-six animals were studied in three experimental groups of 12 animals each. Each group was divided into subgroups and microcirculatory measurements were taken at two different time periods: 24 and 72 hours. In the ischemic control group (N = 12), cremaster muscles were denervated, prepared as a tube flap, and submitted to the same interval of ischemia as the other groups but without transplantation. In the isograft group (N = 12), rat hind limb-cremaster grafts were transplanted between genetically identical Lewis rats (RT11). In the allograft group (N = 12), 12 transplantations were performed across a major histocompatibility barrier between Lewis Brown-Norway (RT-11+/-n) and Lewis (RT 11) rats. The diameters of first-, second-, and third-order arterioles and venules; red blood cell velocities; and functional capillary density were recorded at 24 and 72 hours after transplantation. Daily follow-up observations were continued until 3 days after the first clinical signs of graft rejection. The mean number of perfused capillaries in the two transplantation groups was significantly lower than in the control group at both 24 hours and 72 hours (p < 0.05). Those results were as follows: 8.2 +/- 2.1 at 24 hours, 7.7 +/- 0.85 at 72 hours in the ischemic control group; 5.4 +/- 0.9 at 24 hours, 6 +/- 0.6 at 72 hours in the isograft group; and 5 +/- 0.9 at 24 hours, 5.5 +/- 0.3 at 72 hours in the allograft group. Red blood cell velocities and vessel diameters in the main arteries were also decreased in transplant groups at 24 hours (p < 0.05) but returned to normal 72 hours after the operation (p > 0.05). The composite rat hind limb-cremaster model presented in this study introduces a reproducible in vivo approach to monitor the differences in microcirculatory hemodynamics of ischemia/reperfusion injury and acute graft rejection. The model allows the study of the timing, sequence, and correlation between clinical and hemodynamic signs during the acute phase of allograft rejection.

Effects of 8-Gy radiation on the microcirculation of muscle flaps in the rat.

Combination of radical excision and radiation has been used as a treatment modality for cancer patients. As a result, in reconstructive surgery there is often a need to harvest flaps in the vicinity of previously irradiated tissues. Radiation has been shown to cause progressive injury to the macrocirculation and microcirculation, often jeopardizing flap survival. The purpose of this study was to examine whether radiation significantly affects the sequence of leukocyte-endothelial interactions or the hemodynamics of the muscle flap in both acute and chronic situations. Male Sprague-Dawley rats (n = 42) were divided into seven groups of six rats each. Rats in group I were not irradiated. Groups II through VII received 8-Gy radiation to the right groin and scrotum. Groups II, III, and IV were examined at 4, 24 and 72 hours, respectively, and groups V, VI, and VII were examined at 1, 2 and 12 weeks. For intravital microscopy, the cremaster muscle was dissected on its neurovascular pedicle. Vessel diameters and red blood cell velocities were measured in the central cremasteric branches and branch arterioles. Capillary perfusion was evaluated in 27 visual fields of each flap. Leukocyte-endothelial interactions were evaluated by numbers of rolling, adhering, and transmigrating leukocytes in post-capillary venules. In the same postcapillary venule, we measured the endothelial edema index (constriction index). The hemodynamics of irradiated flaps did not differ significantly from those of controls. Diameter and red blood cell velocity were increased in the first- and second-order arterioles and were highest at 72 hours and 1 week. After irradiation, third-order arterioles were constricted. Radiation reduced capillary perfusion by 4.3, percent. None of the differences were statistically significant. Neither leukocyte behavior nor the constriction indices differed among the groups. In conclusion, low-dose radiation of 8 Gy does not affect hemodynamics or leukocyte-endothelial interactions of muscle flaps in the rat. Muscle tissue with intact microvasculature can be harvested for reconstructive procedures after low-dose radiation.

Muscle flaps' triphasic microcirculatory response to sympathectomy and denervation.

Whether sympathectomy and somatic denervation in muscle flaps increased microcirculatory flow in the short or long term, thus producing an effect similar to the delay phenomenon, which increases survival in transferred skin flaps, was determined. The rat cremaster muscle flap model was used for in vivo microscopy. In the left cremasters of 30 Sprague-Dawley rats, the genitofemoral nerve was divided and the proximal vessels were stripped of their adventitia. The muscle was not elevated. In each rat, the contralateral cremaster served as the control. The rats were assigned to one of five groups: no delay before observation, a 24-hour delay, a 48-hour delay, a 7-day delay, or a 14-day delay. After the delay, red blood cell velocity, vessel diameters, number of functional capillaries, and leukocyte-endothelial interactions were measured. Microvessel response to topical vasoactive substances was measured. Immediately after denervation, red blood cell velocity increased transiently (71 percent; p = 0.006). Main arterioles dilated (20 percent; p = 0.02) at 24 hours, and capillary perfusion increased 36 percent (p = 0.001) at 2 weeks. The microvessels had hyperactive responses to all vasoactive agents 2 weeks after denervation. These findings indicate that proximal sympathectomy with somatic denervation leads to a triphasic, dynamic response in the peripheral microcirculation of the cremaster muscle flap. An initial acute hyperadrenergic phase was followed by a nonadrenergic phase, with significant vasodilatation, and a sensitized phase, with increased capillary perfusion and hyperresponsiveness to vasoactive substances. This study shows that with minimal access to the cremaster muscle flap neurovascular pedicle and without changing the blood supply to the flap, significant hemodynamic improvements can be made in the peripheral microcirculation.

Determination of hindlimb transplantation-induced vascular albumin leakage and leukocyte activation during the acute phase of rejection.

Following transplantation, the microvascular endothelium and endothelial cells play a critical role in allograft rejection, as well as response to surgical trauma. In this study, endothelial-cell damage was assessed through microvascular permeability, and the role of surgical trauma was evaluated during the acute phase of limb allograft rejection. Eighteen isograft and 18 allograft composite-tissue transplantations were performed between 72 rats. At 24-hr, 72-hr, and 7-days follow-up, microvascular permeability, leukocyte activation, functional capillary perfusion, red-blood-cell velocity, vessel diameter, and an endothelial edema index were measured. The permeability index (PI) was statistically significantly greater in the allografts at all follow-up points, compared with the isograft controls (p <0.001). The number of rolling leukocytes was significantly greater in the allografts at 24 and 72 hr; the number of sticking and transmigrating leukocytes was greater at all three follow-up points; and the number of rolling lymphocytes was greater at 7 days (p <0.05). These findings demonstrate the increased rejection phenomenon in allografts, and the increased susceptibility to ischemia and reperfusion injury, compared with isograft transplants. Increased leukocyte activation and acute destruction of endothelial-cell barrier function were demonstrated during the acute rejection period following composite limb allotransplantation.

Delayed gastric emptying affects outcome of Nissen fundoplication in neurologically impaired children.

BACKGROUND: Nissen fundoplication (NF) has a relatively high failure rate in neurologically impaired children with gastroesophageal reflux (GER). In 1990 we began to use routine technetium 99m sulfur colloid emptying scans and pyloroplasty with NF for delayed gastric emptying (DGE) in our neurologically impaired patients. The aim of this study was to determine the influence of DGE and pyloroplasty on the outcome of NF in neurologically impaired children. METHODS: One hundred neurologically impaired children underwent NF by a single surgeon between August 1986 and July 1995. Beginning in January 1990 emptying scans were routinely obtained, and patients with DGE underwent pyloroplasty with NF. Outcome analysis was performed for recurrence/wrap failure and other parameters. Mean follow-up was 5.8 years, with a minimum of 18 months. RESULTS: DGE was found in 35 (65%) of the 54 children who had emptying scans. All 11 children with normal scans had successful NF without recurrent reflux (100%). Forty (93%) of 43 children who underwent pyloroplasty and NF had successful outcomes. Thirty-eight children underwent NF without evaluation of gastric emptying with success in 30 of them (78.9%). Overall success improved from 34 (83%) of 41 in the first half of the study, when 3 (7%) of 41 children underwent emptying scans, to 55 (93%) of 59 in the second half, when 51 (86%) of 59 of the children underwent emptying scans. CONCLUSIONS: DGE is common in neurologically impaired children with GER. NF in children with normal gastric emptying has a high probability of success. Pyloroplasty improves the outcome of NF in children with DGE. Neurologically impaired children should be evaluated for DGE before operation for GER.

Continuous rat intravenous infusion.

Hypovolemic shock and ischemic injury to the graft commonly cause death in small animals after organ transplantation. A venous line must be readily available to replace fluids before fatal complications occur. To establish a venous line, researchers expose a vein by preliminary surgery. This time-consuming procedure adds unnecessary trauma to the recipient and worsens the results. The possibility of long-term fluid transfusion in small animals by serial injections at close intervals is quite limited. We describe a simple technique of continuous i.v. infusion by catheterization of the rat dorsal penile vein with a 24-gauge, 3/4-inch catheter. This easy-to-learn technique has permitted us to establish a venous line quickly without trauma in 148 rats while doing donor and recipient procedures for small bowel and ileocecal segment transplantation. The technique we describe has eliminated one of the most frequent causes of postoperative mortality after organ transplantation-hypovolemic shock. We would like to emphasize that other measures, including avoiding massive bleeding and reducing operative and warm ischemic time, are also very important in preventing this complication. The massive i.v. infusion alone may not totally eliminate hypovolemic shock if other factors are neglected. The use of this technique has allowed us to perform small bowel transplantation with 90% success.