WO2010097477A2

WO2010097477A2 - Means and methods of preserving grafts

Info

Publication number: WO2010097477A2
Application number: PCT/EP2010/052570
Authority: WO
Inventors: Tanja Herrler; Anne Tischer; Carsten Isert
Original assignee: Ludwig-Maximilians-Universität München
Priority date: 2009-02-27
Filing date: 2010-03-01
Publication date: 2010-09-02
Also published as: WO2010097477A3

Abstract

This invention provides for the use of a biocompatible membrane for closing an incision in a medical graft. Also, a method of preserving function of a medical graft by relieving pressure from the graft by incising the graft is described in the present invention.

Description

MEANS AND METHODS OF PRESERVING GRAFTS

This invention provides the use of a biocompatible membrane for closing an incision in a medical graft. Also, a method of preserving function of a medical graft by relieving pressure from the graft by incising the graft is described in the present invention.

The level of the decrease or even loss of the function, regeneration and survival of a medical graft correlates with the ischemia time the graft is subjected to. Although ischemia is not the only parameter responsible for decreased graft function, it is known to be a major risk factor for chronic allograft nephropathy (Schwarz et al., Kidney Int, 67 (2005), 341 -348). Also, dysfunctions such as rarefaction of peritubular capillaries are known to follow ischemic injury and to represent a critical event that may permanently alter the renal function and predisposes patients to the development of chronic renal insufficiency (Basile, Curr Opin Nephrol Hypertens, 13 (2004), 1 -7). Ischemia is further described in the context of ischemia-reperfusion injury, which is known to be associated with the activation of immune cells, capillary leakiness and oedema formation (Gute et al., MoI Cell Biochem, 179 (1998), 169- 187). Although patients with prolonged cold ischemia time (CIT) may receive adequate immunosuppression to prevent acute rejection occurrence, shortening CIT as far as possible would help to decrease not only delayed graft failure rates, but also acute rejection incidence and, hence, graft loss (Mikhalski et al., Transplantation, 85 (2008), S3-S9).

Furthermore, ischemia (and reperfusion) may lead to a compartment syndrome which mostly occurs in the extremities. Compartment syndromes are characterized by critically impaired microcirculation, interstitial edema and an increase in compartmental pressure, and require immediate treatment. Untreated, this condition may result in permanent neurovascular damage, myoglobinuria, renal failure, sepsis and death (Tuckey, Br J Anaesth, 77 (1996), 546-549). An unresolved issue is the prevention of a decrease of graft function caused during ischemic time and unrelated to allogen-dependent immune response in order to avoid acute rejection and graft loss. Efficient strategies to counter disease progression are still missing.

To address this issue, the inventors of the present application have measured the pressure in a graft following ischemia and investigated the extent and role of edema- related pressure elevation within the renal compartment for the long-term functional outcome of kidney transplants.

Surprisingly, they found that also in a medical graft a compartment syndrome can occur and the pressure in the graft increases proportionally to ischemia time which has not been described before. According to these data, increased pressure within the renal compartment plays a major role in the development of chronic allograft dysfunction via microcirculatory impairment, increased inflammatory response, and direct cell damage. Surgical pressure relief according to the present invention by minimal incision effectively prevents tissue injury and functional loss and represents a promising strategy to improve function and survival of the allograft, in accordance with the present invention, such an incision can then be closed with a biocompatible membrane described herein in order to further improve function and survival of the allograft.

Accordingly, the present invention provides a membrane for use in closing an incision in a medical graft. In one embodiment, the incision has been made in order to relieve pressure from the graft. This invention also provides methods for preserving function of a medical graft by relieving pressure from the graft by incising the graft. The incision can be made at any time, i.e. before, during or after the removal of the graft from the donor and before, during, or after the implantation of the graft into the recipient.

The present invention unravels the influence of increased pressure within a graft, e.g., the renal capsule, on the functional outcome with regard to a surgical treatment option. In a murine model of ischemia-reperfusion injury that regularly leads to marked impairment of kidney function and blood flow as well as atrophy, the present invention demonstrates the efficacy of capsulotomy to significantly preserve both functional parameters and structural integrity of the kidney. Importantly, only minimal incision is necessary to confer renoprotection and beneficial effects are evident even in long-term follow-up. These findings explain the role of intracapsular pressure increase in the development of postischemic kidney dysfunction.

In the present invention, it was found that prolonged ischemia was associated with an early edema-related pressure increase within the first 24 h post ischemia and, strikingly, a late de novo rise in pressure on day 18; see Example 2. The latter may be attributed to contraction due to atrophy and fibrosis. Considerably dilated tubules exhibit the histological correlative of pressure increase at 6 h post ischemia, whereas interstitial edema appears to play a major role at 12 and 24 hours reperfusion time. Mild ischemia results in early moderate pressure increase, but has no consequences on intracapsular pressure, renal function and blood flow in the long term. In contrast, chronically elevated pressure involved persistent impairment of renal function and perfusion. The sudden and early decline of the renal excretion rate following prolonged ischemia is possibly the result of an initial no-refiow phenomenon following excessive renal ischemia. In this respect, it is important to know that once a compartment syndrome has developed, it is aggravated by a reinforcing vicious cycle, as increased compartmental pressure is further enhanced by impaired blood circulation. Thus, incision of the kidney capsule in accordance with the present invention prevents irreversible impairment of vascular perfusion after prolonged ischemia and allows considerable functional restoration.

In accordance with the present invention, a closure of the incision using a biocompatible membrane may limit complications for the following reasons: An intact limit of the graft prevents bleeding. Also, uncontrolled loss of fluids/ secretion may be avoided, e.g., urine and bile in the case of kidney and liver transplantation, respectively. Accordingly, the present invention provides a membrane for use in closing an incision in a medical graft. In one embodiment, the incision has been made in order to relieve pressure from the graft.

The present invention further relates to the use of the biocompatible membrane as described herein for closing an incision in a graft.

The present invention also provides a method of preserving the biological function of a medical graft by relieving pressure from the graft, comprising the incision of said graft.

In another embodiment, the present invention also provides a method for preserving biological function of a medical graft by relieving pressure from the graft by incising the graft. The incision may be closed any time following the incision. The decision whether to close the incision or not depends on the size of the incisions and on the threat of leakages of blood and/or urine following the incision.

In one embodiment, the incision is closed following the incision. The incision may be closed by using a membrane, a stapler, a suture and/or a glue. The suture may be resorbable or non-resorbable. In one embodiment, the suture is resorbable. The suture may be stretchy and/or lax so as to allow relief of pressure. The glue may be a fibrin glue. Such glue may be commercially obtained, such as Tissucol Duo and Tissucol Kit lmmuno fibrin glue (Baxter), Dermabond (Ethicon), Beriplast P Combi- Set 0,5/- 1 /-3 ml fibrin glue set (CSL Behring/Nycomed), TachoSil (Nycomed), Quixil (OMRIX), Haesmocomplettan (CSL Behring), Tissue adhesive (B.Braun), Tissue wound closure (Edwards), or the like.

The biocompatible membrane used for closing the incision in the medical graft in accordance with the present invention may be a biocompatible membrane. The biocompatible membrane may be micro-perforated, woven or knitted, 2-dimensional or 3-dimensional. The biocompatible membrane may be a collagen biomatrix, a fibrin biomatrix or a synthetic mesh which easily adapts to the organ surface. The synthetic mesh may be polyester, vicryl or ePTFE. Such membranes may be commercially obtained, Gore PRECLUDE® MVP® Dura Substitute, DUALMESH® Plus, RESOLUT ADAPT LT; Vaskutek Geisoft™ Patch, Gelseal™ Patch, Thin Wail Carotis™ Patch, Thin Wall Fluoropassiv™ Patch; Ethicon/Johnson & Johnson Vicryl Membrane (all shapes and sizes); Nycomed TachoSil® (surgical patch); Davol/BARD Bard Composix Bard E/X Mesh and L/P Mesh, Duplex Mesh, Ventralex Patch, Supramesh IP Composite; Collamend Implantat, AlloMax Surgical graft, Bard CruraSoft Patch; Cook Medical Biodesign Advanced Tissue Repair; JOTEC flowline biopore; B.Braun Optilene Mesh, Optilene Mesh LP, Optilene Mesh elastic, Premilene, Safil Mesh, Safil Mesh bag; Baxter Tissue Dura, TissueFoil E; Covidien Alginates, AMD™ Antimicrobial Dressings, Foam Dressings, Hydrocolloid Dressings, Hydrogels, Island Dressings, Thermazene™ Cream (1 % Silver Sulfadiazine Cream), Traditional Wound Care. Transparent Film Dressings, Permacol; Lamed Peri-Strips Dry; Tutogen Tutoplast®; Tissue Science Laboratories Permacol Surgical Implant or the like. The membrane may be flexible or rigid. The membrane may be permeable for pressure. The membrane may also have valve function wherein pressure can penetrate the membrane. The membrane may be self-adhesive. The membrane may also be fixed to the graft in order to close the incision. The fixation of the membrane on the graft may be made using a stapler, a suture or glue. The suture may be resorbable or non- resorbable. In one embodiment, the suture is resorbable. The suture may be stretchy and/or lax so as to allow relief of pressure. In another embodiment, the membrane is fixed using a glue. The glue may be a fibrin glue. Such glue may be commercially obtained, such as Tissucol Duo and Tissucol Kit lmmuno fibrin glue (Baxter), Tisseel (Fibrin sealant) Kit with Duploject System 2 ml (Baxter), Dermabond (Ethicon), Beriplast P Combi-Set 0.5/-1/-3 ml fibrin glue set (CSL Behring/Nycomed), TachoSil (Nycomed), Quixil (OMRIX), Haesmocomplettan (CSL Behring), Tissue adhesive (B.Braun), Tissue wound closure (Edwards), or the like. The membrane may also be coated with one or more active compounds to enhance regeneration or to prevent damage from the graft. Such compounds may be cytokines. In one embodiment, the compound is VEGF (vascular endothelial growth factor). In another preferred embodiment, the compound is an immunosuppressive and/or anti-inflammatory agent. The membrane may be of any size. It may be round, oval or any other shape suited for application to the graft. In one embodiment, the membrane has the size and/or shape of the incision plus a marginal addition sufficient for safe fixation of the membrane on the intact tissue. That is, depending on the size of incision, the membrane must cover the area of the incision and additional area of the graft for fixation. The additional area may be about 2 mm to about 20 mm. Preferably, the additional area is about 5 mm to about 15 mm, most preferably about 10 mm. Furthermore, the upper side of the biocompatible membrane of the present invention is preferably non-adhesive to avoid adhesion of the graft to surrounding tissue. For example, the membrane may be coated with a silicone or any other biocompatible non-adhesive compound.

In accordance with the present invention, the biocompatible membrane may exhibit the following characteristics in addition to the properties described hereinabove: The biocompatible membrane of the present invention may be initially permeable. The membrane may exhibit permeability for fluids of any kind (e.g., blood, urine or intestinal fluid for about 7 days after being attached onto the draft to close the incision in order to allow drainage of interstitial fluid. For example, the membrane may be permeable for 5, 6, 7, 8 or 9 days. Subsequently, the biocompatible membrane according to the present invention becomes fluid-impermeable.

In accordance with the present invention, the incision can be made at any spot of the graft suitable to relief pressure from the inner graft. If the graft is a kidney, the incision is preferably done at the lower pole of the kidney; see Figure 8. The incision may be of any size. The size depends on the size of the organ. The incision may be round or linear. In one embodiment, the incision is round. The incision may also be a puncture or a cut. In a preferred embodiment, the incision is round, e.g., a puncture. In accordance with the present invention, a round incision is preferably about 3 mm to about 15 mm in diameter, most preferably about 5 mm to about 7.5 mm. If the incision is a cut, the length of the incision is preferably about 5 mm to about 30 mm, most preferably about 10 mm to about 20 mm. The incision may be made with a needle, a scalpel or any other means suitable. The incision may be made at any site of the graft. In one embodiment, the incision is made in the renal capsule in a kidney. The incision may be made any time before, during or after the removal of the graft from the donor. In one embodiment, the incision is made after the removal of the graft from the donor. The incision may be made any time before, during or after implantation of the graft into the recipient. In one embodiment, the incision is made before the implantation of the graft into the recipient, i.e. before the reperfusion period. There may also be made more than one incision of the same or different type.

The incision may be closed using the membrane in accordance with the present invention any time following the incision. The best point of time for closing the incision depends on the occurrence of bleedings and/or leakage of urine. In one embodiment, if an incision is given at this point of time, the incision is closed before the implantation of the graft into the recipient, i.e. before the reperfusion period. After the closure of the incision, the closure may be checked for leakages of blood or urine.

In accordance with the present invention, the donor and/or the recipient of the graft may be mammal. In one embodiment, the recipient is human. The donor may be alive or deceased at the time the graft is removed. In one embodiment, the donor is deceased at the time the graft is removed. The graft may be an autograft, an allograft or a xenograft. In one embodiment, the graft is an allograft. The graft may be a kidney, heart, lung, liver, pancreas, intestine, hand, foot, arm, leg, digit, toe, cornea, skin, penis, Islet of Langerhans, bone, free flaps, or parts thereof. In a preferred embodiment, the graft is a kidney.

The present invention is also suitable to unravel the pathomechanisms of tissue injury following renal ischemia-reperfusion injury and a surgical therapeutic approach in animal models, e.g., the swine model. The pathophysiologic role of post-ischemic pressure increase within the kidney capsule can be determined. Furthermore, the renoprotective effect of a surgical incision of the kidney capsule for pressure relief can be evidenced. In the Examples described herein, the efficacy of this treatment in a murine model of renal ischemia-reperfusion injury is demonstrated. With regard to clinical application, the effect of surgical decompression can be examined in a large animal model (e.g., swine) to confirm the applicability in humans. The data provided herein in the Examples from the murine model demonstrate that pressure relief for renoprotection is essential during the acute phase following ischemic injury. In order to prevent complications due to the therapeutic incision, i.e. formation of lymphoceles, urinoma, and bleeding, the therapeutic application of a biocompatible membrane for closure of the incision can be demonstrated. As already described above, membrane characteristics according to the present invention include initial permeability and spontaneous closure of pores after the acute phase.

Brief description of the Figures

Figure 1 : Measurement of subcapsular pressure in murine kidney during 35 min and 45 min ischemia time: Subcapsular pressure was continuously measured using a Codman ICP microsensor (Johnson & Johnson Medical Limited, Berkshire, UK). Mice were anesthetized and placed on a heated surgical pad to keep constant body temperature. The right kidney was exposed through median abdominal incision. The ICP probe was advanced 2 mm under the kidney capsule through a minimal incision. Subcapsular pressure was continuously monitored for 10 min. The probe was removed and the kidney harvested for histological analysis.

Figure 2: Histological pictures of renal tissue corresponding to the measurements shown in Figure 1 after 45 min ischemia: Tissue sections show murine renal cortex subjected to 45 min ischemia at 6h (A), 12h (B), 24h (C), 48h (D), and 18 days (E) after reperfusion, original magnification was x200. Depending on ischemia time, kidneys exhibited severe tissue damage including cytoplasmic degeneration, excessive tubular atrophy / necrosis, oedema, and fibrosis. Oedema is predominating at 12 h reperfusion time. 24 h hours post ischemia many protein cylinders were found, whereas following 48 h the extent of oedema and protein cylinders is decreased. Long-term follow-up is characterized by vast tissue necrosis, fibrosis, and calcification.

Figure 3: Prolonged ischemia-reperfusion injury leads to irreversible loss of tubular excretion function that is effectively prevented by incision of the kidney capsule. (A) After 45 min ischemia, scintigraphy revealed early marked impairment of tubular excretion function and renal function peak in reference to the healthy kidney with no evidence for spontaneous restoration in the long term. (B) Shorter ischemia time of 35 min was not associated with a significant decline in renal function peak and excretion rate. (C) Surgical pressure relief by surgical incision was able to significantly prevent loss of renal function after prolonged renal ischemia of 45 min. (D) Renal function peak was only reduced by 20% in the incision-treated kidneys vs. almost 60% reduction in the nontherapy group. (E) Tubular excretion rate in the therapy group was significantly higher than in nontreated animals. %ID, percentage injected dose. Pre, baseline renal function before surgery.

Figure 4: Laser Doppler assessed perfusion measurement. No change in vascular perfusion was recorded 18 days after induction of ischemia- reperfusion injury for treated ischemic kidneys, whereas prolonged ischemia time (45 min) resulted in significant impairment of renal perfusion.

Figure 5: Kidney size and weight following ischemia-repersfusion injury. Excessive ischemia-reperfusion induces irreversible tissue damage and atrophy. (A) In reference to their healthy counterpart, kidneys exposed to prolonged ischemia time of 45 min showed considerably more reduction in size than incision-treated kidneys. (B) Consistently, nontreated ischemic kidneys exhibited marked loss in weight, whereas only moderate weight loss was seen in ischemic kidneys with capsular incision.

Figure 6: Surgical therapy by incision of the kidney capsule prevents tubular necrosis after prolonged ischemia. In reference to the healthy kidney, kidneys subjected to 45 min of ischemia exhibited extensive tissue damage. No distinct changes except for hyperemia were found within the glomeruli. Kidneys exposed to prolonged ischemia but treated by surgical incision presented completely preserved renal structures and excellent viability with no signs for necrosis. Original magnification was x200.

Figure 7: Assessment of renal function by scintigraphy. (A) Image files were analyzed by standard manual region of interest (ROI) determination of the whole body, both kidneys including their background regions and the site of injection. (B) Renal function is represented %ID. Renal function peak and the tubular excretion rate reflected by the delta of [peak %ID minus %ID at 10 min examination time] depict the extent of organ failure. %ID, percentage injected dose.

Figure 8: (A) Site and (B) technique of surgical decompression therapy by circular incision of the kidney capsule in a murine model of renal ischernia- reperfusion injury.

The Examples illustrate the invention. The examples as described below may also be carried out using large mammals as test animals, such as swines.

Example 1

Experimental setup using a murine model of renal ischemia-reperfusion injury

Mice are anesthetized by intraperitoneal injection with a combination of ketamine (150 mg/kg) and xylazine (15 mg/kg) and placed on a heated surgical pad to keep constant body temperature. The right kidney is exposed through median abdominal incision, and mice are subjected to ischemia by clamping the renal pedicle with a non-traumatic microaneurysm clamp (Braun, Melsungen, Germany), which is removed after 35 min or 45 min. The incision is closed with a 5-0 suture and surgical staples. At 6 h, 12 h, 24 h, 48 h, and 18 day reperfusion time, subcapsular pressure is determined and the kidneys harvested for histological analysis (see Figures 1 and 2). Prolonged renal ischemia is associated with a significant rise in subcapsular pressure already after 6 h reperfusion. Pressure values continue to be unphysiologically high until returning to baseline by 48 h post ischemia with a surprising de novo rise in pressure found at 18 day reperfusion. The reason for the de novo increase in pressure in long-term follow-up examination is not known. In contrast, shorter ischemia times do not lead to a significant increase in subcapsular pressure. Consistently, only mild changes in histological appearance were found with tubules showing granular degeneration within the cytoplasm, but no signs of necrosis.

Example 2

Pressure relief in renal graft by incision of the kidney capsule

Animals.

Male Balb/C wildtype mice, aged 10 to 12 weeks and weighing 22-24 g were purchased from Charles River Laboratories (Sulzfeld, Germany). Animals were fed a standard diet and allowed free access to water. All animal experiments were conducted in accordance with institutional guidelines and were approved by the Administrative Panel on Laboratory Animal Care.

Renal ischemia-reperfusion injury model and capsulotomy.

Mice were anesthetized by intraperitoneal injection with a combination of ketamine (150 mg/kg) and xylazine (15 mg/kg) and placed on a heated surgical pad to keep constant body temperature. The right kidney was exposed through median abdominal incision, and mice were subjected to ischemia by clamping the right renal pedicle with a non-traumatic microaneurysm clamp (Braun, Melsungen, Germany) which was removed after 35 min and 45 min, respectively. In the therapy group, the capsule of the right ischemic kidney was punctured at the front side of the lower pole using a 30-gauge needle. The abdominal incision was closed with a 5-0 suture and surgical staples. Intracapsular pressure measurement.

Intracapsular pressure was continuously measured using a Codman ICP microsensor (Johnson & Johnson Medical Limited, Berkshire, UK). Mice were anesthetized by intraperitoneal injection and placed on a heated surgical pad to keep constant body temperature. The right kidney was exposed through median abdominal incision. After minimal incision of the kidney capsule, the ICP probe was advanced 2 mm under the capsule. Intracapsular pressure was continuously monitored for 10 minutes. The probe was then removed and the ischemic kidneys were collected for histological analysis.

Renal scintigraphy using ^99mTechnetium-Mercapto-Acetyl-tri-Glycine ("^mTc-MAG3). Scintigraphy using ^99mTc-MAG3 (Technescan MAG3, Covidien, Neustadt/Donau, Germany), a radioactive compound predominantly excreted by tubular secretion, was performed in analogy to a previously described protocol.⁹ Briefly, after hydration with sterile saline and induction of anesthesia with a combination of ketamine (150 mg/kg) and xylazine (15 mg/kg) mice underwent whole-body scintigraphy in a triple-headed gamma camera (Philips - former Picker - Prism 3000 XP, Cleveland, USA) using dynamic imaging protocols with ^99mTc-MAG3. Each detector head was equipped with a LEHR collimator, but only one head was used. Intravenous injection of a standardized dose of -3.7x10⁷ Bq per mouse and acquisition in a dynamic planar technique were simultaneously started. 1 frame per 5 sec was collected with a total scan time amounting to 10 min. The image acquisition magnification was set to 4 times. To determine baseline renal function, ^99mTc-MAG3 imaging was carried out 4 days before ischemia-reperfusion surgery. Postoperative renal scans were performed on day 2 and 18 for the assessment of early and long-term kidney function. In order to minimize experimental stress due to surgery, day 2 was chosen as the earliest postoperative time point for renal scintigraphy.

Image analysis.

Image files were analyzed using Hermes kidney analysis software V4.1 (Hermes Medical Solution, Stockholm, Sweden) by standard manual region of interest (ROI) analyses of the whole body, both kidneys including their background regions as wells as the site of injection (Fig. 7A). Data were exported to Microsoft Excel to assess renal function represented as percentage of injected dose (%ID). Values for %ID were obtained by division of the background corrected kidney ROI by the injection site corrected whole body ROI. In addition to renal function curves (%ID), peak (%ID) and renal excretion capacity reflected by the delta of [peak (%ID) minus %ID at 10 min examination time] were determined (Fig. 7B).

Renal perfusion measurements.

After final ^99mTc-MAG3 imaging on day 18, blood flow of the ischemic (right) and nonischemic (left) kidney was assessed using the O2C laser Doppler blood flow analyzer with the LFM-2 micro probe (2 mm tissue penetration; both Lea Medizintechnik, Giessen, Germany). Relative perfusion was calculated as the ratio of blood flow in the ischemic and the healthy kidney

Weight analysis.

Before embedding for histological analysis, both the right ischemic kidney and its healthy contralateral counterpart were weighed using a special accuracy weighing machine (Sartorius BP3105, Goettingen, Germany). Considering interindividual differences, kidney weight was expressed as the ratio of the ischemic and the healthy kidney, as similar weight was recorded for both kidneys in an individual.

Histological analysis.

Tissue specimens (ischemia-reperfusion injured right kidney and contralateral healthy kidney) were collected for histological analysis. After paraffin embedding, histological sections were stained with H&E.

Statistical Analysis.

Statictical analysis was performed using paired or unpaired t-test (two-sided). Data are expressed as mean ± s.e.m. P values less than 0.05 were considered statistically significant. RESULTS

Ischemia-reperfusion injury is associated with increased pressure within the renal compartment.

Following ischemia-reperfusion injury, a significant increase in pressure values within the renal compartment in an ischemia time-dependent manner was found (Fig. 1 ). Overall, prolonged ischemia time of 45 min resulted in significantly higher pressure values compared to mild ischemia of 35 min 6 h and 12 h post ischemia (p<0.05). Compared to baseline (0.9 mmHg +/- 0.3 mmHg; n=5), 45 min ischemia led to a 7.6- fold increase 6 h after reperfusion (7.0 mmHg +/- 1.0 mmHg; p<0.001 ; n=5), whereas only a 3.6-fold increase was found for 35 min ischemia time (3.3 mmHg +/ 0.0 mmHg; p<0.01 ; n=5). In the 35 min ischemia group, pressure already declined to baseline at 24 h reperfusion time (2.2 mmHg +/- 0.5 mmHg; n.s. v.s. baseline; n=4). In contrast, pressure values in the 45 min ischemia group continued to be unphysiologically high until returning to baseline by 48h post ischemia (2.7 mmHg +/- 1.0 mmHg; n.s. vs. baseline; n=5). Strikingly, we found a de novo 5-fold increase in pressure during long-term follow-up examination day 18 (4.7 mmHg +/- 1.5 mmHg; p<0.05; n=5).

Prolonged ischemia-reperfusion injury leads to irreversible loss of tubular excretion function.

Renal function curves obtained from ^99mTc-MAG3 imaging were evaluated for peak (maximum uptake) and tubular excretion rate (peak [%ID] minus [%ID] at 10min examination time). Following 45 min ischemia, scintigraphy revealed early marked impairment of tubular excretion function in reference to the healthy kidney (pre/ baseline = 100%) with no evidence for spontaneous restoration in the long term (35.4% +/- 2.6% baseline on day 2; 33.0% +/- 3.5% baseline on day 18; p<0.001 ; n=5). Renal function peak was also considerably reduced on day 2 (79.2% +/- 3.4% baseline; p<0.001 ; n=5) and further declined during subsequent measurements on day 18 (41.6% +/- 3.2% baseline; p<0.001 ; n=5; Fig. 3A). In contrast, no significant decline in renal function peak (94.7% +/- 6.7% on day 2, 95.3% +/- 3.9% on day 18; n.s. vs. baseline; n=7) and excretion rate (59.0% +/- 7.0% on day 2, 99.9% +/- 5.2% on day 18; n.s. vs. baseline; n=7) was recorded following mild ischemia of 35 mm. (Fig 3B).

Incision of the kidney capsule preserves renal function following prolonged ischemia. Incision of the kidney capsule effectively preserved renal function as assessed by scintigraphy following prolonged renal ischemia of 45 min (Fig. 3C). Surgical pressure relief was able to significantly prevent loss of renal function. In contrast to the control group (peak: 41.6% baseline +/- 3.2%), renal function peak was only reduced by 20% (peak: 80.2% baseline +/- 10.7%; p<0.05 vs. control; Fig. 3D). Tubular excretion rate in the therapy group was as high as 62.5% baseline +/- 6.8% compared to 33.0% baseline +/- 3.5% for control animals (p<0.01 ; Fig. 3E).

Irreversible impairment of vascular perfusion after prolonged ischemia is prevented by incision of the kidney capsule.

No significant change in vascular perfusion was recorded 18 days after induction of ischemia-reperfusion injury for animals undergoing 35 min of renal ischemia as assessed by laser Doppler (99.5% +/- 1.5%; n.s. vs. healthy; n=7). In contrast, prolonged ischemia time of 45 min led to significant impairment of renal perfusion (64.5% +/- 6.8% healthy; p<0.005; n=5). Incision of the kidney capsule significantly preserved renal blood flow (96.2% +/- 4.8%; p<0.05 vs. control, n.s. vs. healthy; n=8; Fig. 4).

Excessive ischemia-reperfusion induces irreversible tissue damage and atrophy. In reference to their healthy counterpart, kidney exposed to prolonged ischemia time of 45 min showed considerably more reduction in size than incision-treated kidneys (Fig. 5A). Non-treated kidneys exposed to ischemia exhibited marked loss in weight (66.6% healthy +/- 8.0%; p<0.05 vs. healthy; n=5) than kidneys with capsular incision (87.3% healthy +/- 7.9%; p<0.01 vs. control, n.s. vs. healthy; n=8; Fig. 5B).

Surgical therapy by incision of the kidney capsule prevents tubular necrosis following prolonged ischemia.

Following renal ischemia time of 45 min, the extent of interstitial edema, varied depending on the time of examination. Whereas histology was characterized by markedly dilated tubules at 6 h reperfusion time and only slight interstitial edema, pronounced interstitial edema was seen 12 h and 24 h after ischemia. At 48 h post ischemia edema was considerably reduced (Fig. 6A). In reference to the healthy kidney, the extent of tissue damage correlated with increasing ischemia time. On day 18 after induction of ischemia-reperfusion injury, 35 min of ischemia resulted in only mild changes in histological appearance including dilated tubules, granular degeneration within the cytoplasm, and without any signs of necrosis. Glomeruli as well as brush borders were well preserved. In contrast, kidneys subjected to 45 min of ischemia exhibited extensive tissue damage including tubular atrophy and necrosis, excessive dilatation and cytoplasmic degeneration, loss of brush borders, protein cylinders, and multiple areas of calcification within the cortex as a consequence of previously occurred tubular necrosis. No distinct changes except for hyperemia were found within the glomeruli. Kidneys exposed to prolonged ischemia, but treated by surgical incision presented completely preserved renal structures and excellent viability with no signs for necrosis (Fig. 6B).

Example 3

Medical procedure

Explantation of kidney transplant according to established protocol from living or cadaveric donor, the latter being more at risk for postoperative allograft dysfunction. Donor and recipient are both human. Two round incisions amounting to 5 mm diameter using a scalpel are made on the front side of the kidney, one at the upper and one at the lower pole, during preparation of the allograft for implantation while soaked in cold preservation solution. Incisions are immediately closed by a biocompatible pressure-compensating membrane that was trimmed to overlap the margin of the incision in the renal capsule (app. 10 mm) and is attached using fibrin glue. The transplant kidney is implanted and reperfused according to established standard and checked for permeability of the membranes. Then, the wound is closed.

Claims

1. Biocompatible membrane for use in closing an incision in a medical graft.

2. Method of preserving the biological function of a medical graft by relieving pressure from the graft, comprising the incision of said graft.

3. Membrane according to claim 1 or method according to claim 2, wherein the incision is made after the removal of the graft from the donor and before implantation of the graft into the recipient,

4. Membrane according to claims 1 or 3 or method according to claim 2 or 3, wherein the donor and the recipient is mammal.

5. Membrane according to any one of claims 1 , 3 or 4 or method according to any one of claims 2 to 4, wherein the incision is round, punctual or linear.

6. Membrane or method according to claim 5, wherein said round incision is about 3 to about 15 mm, preferably about 5 mm to about 7.5 mm in diameter.

7. Membrane or method according to claim 5, wherein said linear incision is about 5 to about 30 mm, preferably about 10 mm to about 20 mm in length.

8. Method according to claims 2 to 7, wherein the incision is closed following the incision.

9. Method according to claim 8, wherein the incision is closed with a biocompatible membrane, a suture and/or a glue.

10. Membrane according to any one of claims 1 or 3 to 7 or method according to claim 9, wherein the membrane is flexible.

1 1. Membrane according to any one of claims 1 , 3 to 7 or 10 or method according to claim 9 or 10, wherein the membrane has a valve function for pressure relief.

12. Membrane according to any one of claims 1 , 3 to 7 or 10 or method according to claim 9 or 10, wherein the membrane is initially permeable.

13. Membrane according to any one of claims 1 , 3 to 7, or 10 to 12 or method according to any one of claims 9 to 12, wherein the membrane is fixed with a suture or a glue.

14. Membrane according to any one of claims 1 , 3 to 7 or 10 to 13 or method according to any one of claims 9 to 13, wherein the membrane is coated with one or more active compounds to enhance regeneration or to prevent damage from the graft.

15. Membrane or method according to claim 14, wherein the active compound is one or more cytokine, an immunosuppressive and/or anti-inflammatory agent.

16. Membrane or method according to claim 15, wherein the active compound is VEGF.

17. Membrane according to any one of claims 1 , 3 to 7 or 10 to 16 or method according to any one of claims 2 to 16, wherein more than one incision is made.

18. Membrane according to any one of claims 1 , 3 to 7 or 10 to 17 or method according to any one of claims 2 to 17, wherein the graft is autograft, allograft or xenograft.

19. Membrane according to any one of claims 1 , 3 to 7 or 10 to 18 or method according to any one of claims 2 to 18, wherein the graft is kidney, heart, lung, liver, pancreas, intestine, hand, foot, arm, leg, digit, toe, cornea, skin, penis, Islet of Langerhans, bone, free flaps, or parts thereof,

20. Use of the membrane according to any one of claims 1 , 3 to 7, or 10 to 19 for closing an incision in a graft.