S. Simpkin

REDUCED SUSCEPTIBILITY TO LIPID-PEROXIDATION IN COLD ISCHEMIC RABBIT KIDNEYS AFTER ADDITION OF DESFERRIOXAMINE, MANNITOL, OR URIC-ACID TO THE FLUSH SOLUTION

Rabbit kidneys were stored for 24 hr at 0 degree C after single passage arterial flush with 30 ml of cold isotonic 0.9% sodium chloride (saline) solution alone or saline to which was added 12, 30, or 60 mM desferrioxamine, 1 or 3 mM uric acid, or 100 mM mannitol. They were then subjected to in vitro biochemical assay for evidence of free radical damage immediately after storage. Results were compared to those obtained with fresh, unstored kidneys. Levels of Schiff base fluorescence, diene conjugates, and thiobarbituric acid-reactive material were each significantly elevated in kidneys stored for 24 hr after flush with saline alone. These levels were in turn each significantly reduced by the addition of 60 mM desferrioxamine, 3 mM uric acid, and 100 mM mannitol to the flush solution. Likewise, glutathione redox activity fell in those flushed with saline alone, presumably in line with increased lipid peroxidation, but was restored to control levels by inclusion of the three scavenging agents.

Desferrioxamine reduces susceptibility to lipid peroxidation in rabbit kidneys subjected to ward ischaemia and reperfusion

Rabbit kidneys were clamped and subjected to warm ischaemia for 60 or 120 min then reperfused with blood for 60 min or for 24 hr. Treated rabbits received desferrioxamine at 15 or 50 mg/kg i.v. 15 min before reperfusion. Their kidneys were then removed and assayed for phospholipid Schiff base fluorescence (ex. 360 nm, em. 435 nm), diene and triene conjugates by UV spectrophotometry (240 nm and 268 nm respectively), for superoxide dismutase and for reduced and oxidised glutathione to provide an index of glutathione redox activity. All indices of lipid peroxidation were significantly elevated in untreated rabbits and glutathione redox activity was reduced. Treatment with desferrioxamine however effectively prevented these deviations and in many cases maintained them at the levels in fresh rabbit kidneys. These data provide further evidence that lipid peroxidation occurring during the reperfusion period is superimposed on the damage set up during warm ischaemia and may be preventable by administration of suitable therapeutic agents.

The Importance of Iron, Calcium and Free Radicals in Reperfusion Injury: An Overview of Studies in Ischaemic Rabbit Kidneys

An overview of a series of experiments attempting to link iron and calcium redistribution and release of free fatty acids with falls in pH and adenine nucleotide levels during cold storage of rabbit kidneys is presented. The data reviewed strongly suggest that these events are inextricably linked to subsequent reperfusion injury. Circumstantial evidence incriminating iron was provided by experiments showing that iron chelation decreased reperfusion injury after warm (WI) and cold ischaemia (CI) in rat skin flap and rabbit kidney models. Evidence for a role for calcium was provided when it was found that a calcium channel blocking agent added to the saline flush solution before storage inhibited lipid peroxidation, whereas chemicals which caused release or influx of calcium into the cell exacerbated oxidative damage. Additional involvement of breakdown products of adenine nucleotides was suggested by the protection from lipid peroxidation afforded by allopurinol. Involvement of calcium-activated phospholipase A2 was strongly suggested by increases in free fatty acids during cold storage and both this increase and lipid peroxidation were inhibited by addition of dibucaine to the storage solution.

Protection against oxidative damage in cold-stored rabbit kidneys by desferrioxamine and indomethacin

The storage of rabbit kidneys in hypertonic citrate solution at 0 degree C for 48-72 hr of cold ischemia resulted in oxidative damage to membranes as measured by the in vitro formation of two markers of lipid peroxidation (Schiffs base and thiobarbituric acid (TBA)-reactive material). This damage was further increased when the organs were autografted and reperfused for 60 min. The intravenous (iv) administration of desferrioxamine (a powerful iron-chelating agent) prior to the removal of the kidneys reduced the production of Schiffs bases and TBA-reactive material to low levels in the cortex of stored kidneys and decreased these measures of lipid peroxidation in the medulla by approximately 50%. Intravenous administration of indomethacin (a cyclooxygenase inhibitor) had no effect on the rate of lipid peroxidation in the renal cortex, but significantly reduced the formation of TBA-reactive material and Schiffs bases in the medulla of kidneys following storage for 72 hr. The existence of two separate pathways of lipid peroxidation (one iron-catalyzed and the other cyclooxygenase-catalyzed) in the medulla of stored kidneys was further confirmed when administration of desferrioxamine and indomethacin together resulted in significantly greater protection against lipid peroxidation than when these compounds were administered singly. The value of this combination of agents for protecting kidneys against the damage due to cold ischemia followed by reperfusion was further suggested by a trend toward improved long-term survival of the animals following replantation of the stored kidneys.

Increased susceptibility to lipid peroxidation in rabbit kidneys: A consequence of warm ischaemia and subsequent reperfusion

Rabbit kidneys were clamped and rendered warm ischaemic (WI) in situ for 60 and 120 min. They were then either removed immediately after the ischaemic insult or after reperfusion with blood for 60 min or 24 hr. Homogenates were assayed for phospholipid-Schiff base fluorescence (Ex. 360 nm, Em. 435 nm) and for diene conjugate formation by u.v. spectrophotometry (240 nm) as indices of lipid peroxidation. No alteration in tissue levels of Schiff base was evident immediately after WI but when the homogenates were incubated at 37 degrees C for 90 min, the rate of peroxidation was significantly elevated compared to controls (P less than 0.02 after WI of 60 min and P less than 0.001 after 120 min of WI). These values were still further elevated after reperfusion with blood for 60 min and 24 hr (P less than 0.001). Diene conjugates were raised after WI alone and further still after reperfusion. Thus an early index of lipid peroxidation (diene conjugation) suggested peroxidative damage during the warm ischaemic period itself, whilst detection of Schiff bases was only possible after in vitro incubation of the tissue. Both indices of oxygen-derived free radical damage were increased after reperfusion in vivo with blood and may relate to the degree of tissue damage sustained during ischaemia and reflow.

Allopurinol Inhibits Lipid Peroxidation in Warm Ischaemic and Reperfused Rabbit Kidneys

Rabbit kidneys were subjected to 120 min of warm ischaemia or to 120 min of warm ischaemia followed by 60 min reperfusion with blood in vivo before being removed, homogenised and incubated at 37 degrees C for 90 min. Lipid extracts were obtained and monitored for Schiff base (fluorescence emission 400-450 nm, excited at 360 nm), thiobarbituric acid (TBA)-reactive material (emission 553 nm, excited at 515 nm) and diene conjugates (absorbance at 237 nm). Samples removed before incubation were assayed for reduced glutathione (GSH) and oxidised glutathione (GSSG) to provide an index of glutathione redox activity (GSH:GSSG). Allopurinol injected systemically i.v. (a) 15 mins before kidneys were clamped, (b) 15 mins before they were reperfused or (c) as two injections (before clamping and before reperfusion) significantly inhibited these biochemical markers of lipid peroxidation. Administration before reperfusion had a markedly more pronounced effect than when allopurinol was given before warm ischaemia only. It is concluded that allopurinol is probably effective because of its ability to inhibit xanthine oxidase and consequently lipid peroxidation during reperfusion rather than by preventing loss of purine nucleotides from hypoxic cells during ischaemia.

Monitoring of Mitochondrial Nadh Levels by Surface Fluorimetry as an Indication of Ischaemia During Hepatic and Renal Transplantation

One of the major causes of dysfunction in transplanted organs is ischaemia-reperfusion (IR) injury. Impairment of mitochondrial function is likely to be central to many of the known consequences of ischaemia; these include loss of cellular homeostasis involving a fall in intracellular pH (Fuller et al., 1988), mitochondrial calcium loading and cellular swelling (Caiman et al., 1973), accumulation of reduced pyridine nucleotides, inhibition of mitochondrial electron transfer, and a fall in ATP levels (Hardy et al., 1991). In irreversibly damaged cells, respiratory control is lost and is accompanied by oxidation of cytochromes a and a3 and NADH (Taegtmeyer et al., 1985). The latter was attributed originally to substrate deficiency (Chance and Williams, 1955) but more recent studies indicate that an enzymological defect develops resulting in an inability to metabolise NADH-linked substrates (Taegtmeyer et al., 1985 and Hardy et al., 1991). In vitro studies of the respiratory chain (RC) complexes have been made in several tissues including cardiac and renal tissue, subjected to ischaemia-reperfusion injury and it was found that complexes I and IV are major defective sites (Hardy et al., 1991 and Veitch et al., 1992). Return of function may, therefore, relate to preservation of inner mitochondrial membrane integrity, and the structure and activities of the RC complexes. The integrity of oxidative metabolic pathways and capacity to resynthesise ATP rather than the immediate post-ischaemic ATP levels appears to determine the return of function (Taegtmeyer et al., 1985).

APPLICATIONS OF NIRS FOR MEASUREMENTS OF TISSUE OXYGENATION AND HAEMODYNAMICS DURING SURGERY

Since 1977 when Jobsis described the first use of near infra-red spectroscopy (NIRS) for non-invasive monitoring of changes in cerebral oxyhaemoglobin (O2Hb), deoxyhaemoglobin (HHb) and Caa3 the field has undergone major transformations1,2.

Measurement of cerebral oxygenation and haemodynamics during haemorrhage/fluid replacement

Many major surgical procedures are accompanied by haemorrhage necessitating replacement with either fluid or blood. Despite apparently adequate replacement, tissue oxygen delivery may still be poor, resulting in cellular hypoxia and consequent metabolic dysfunction, which can ultimately lead to multi-organ failure. The overall aim of these investigations was to determine whether near infra-red spectroscopy (NIRS) can be used to measure changes in cerebral oxygenation and haemodynamics in response to controlled blood loss, with and without fluid replacement using a rat model of haemorrhage1-6.

Reperfusion Injury and Renal Metabolism : The Temporal Relationship Between Oxidative Stress and Functional Change

A large number of studies have implicated oxygen-derived free radical (OFR) stress in the pathology of the so-called ‘reperfusion injury’ in a number of organs and tissues including brain (1), liver (2), intestine (3), skin (4), heart (5) and kidney (6). This is the type of injury sustained when an organ has been deprived of blood supply for a period of time, and is then subjected to a sudden restoration of blood supply bringing in high concentrations of oxygen; such events occur in many surgical interventions e.g. during repair of tissues after trauma, and particularly in organ trans- plantation (7).There is growing evidence that OFR scavengers, when introduced under such circumstances, can beneficially influence the outcome of reperfusion (8). However, there is still much debate about the time relationship between OFR events in a tissue and subsequent functional changes, which in turn will influence the timing of administration of any anti-OFR therapy. We have been particularly concerned with reperfusion injury in kidneys (9,10), and the present studies were undertaken to assess renal tissue metabolism (by glueoneogenesis) in rabbit kidneys after ischaemia / reperfusion. Renal cortical tissue gluconeogenesis was chosen as functional test since this is an active process of cortical tubular cells and these cells show characteristic early signs of ischaemia / reperfusion damage as expressed by tubular necrosis.