Ischemic Effects on Tissues and Organs

Solid organs, such as the heart, liver, kidney and brain, require large amounts of oxygen and fuel to support their various specialized functions. Eighty percent of the oxygen consumed by the human body is used for energy production, which is produced when large, complex molecules (such as carbohydrates, lipids and proteins) are broken down into smaller, simpler molecules. The resultant decrease in chemical order causes the liberation of free energy. It is estimated that in humans, the rate of adenosine triphosphate (ATP) synthesis varies daily from 80 moles (about 40 kg) at rest to 1800 moles (∼ 910 kg) during strenuous exercise.^{2 , 3} In general, glucose metabolism supplies the largest portion of cellular energy through its various steps (i.e., conversion of substrates to acetyl-CoA in the cytoplasm, the oxidation of acetyl-CoA in the Krebs citric acid cycle and electron transfer activities coupled to oxidative rephosphorylation of ADP to ATP in the respiratory chain of the mitochondria, which is responsible for 90-95% of the total ATP requirement in eukaryotic cells).²

A great number of events occur when tissues and organs are removed from the body. As blood circulation stops, there is a combined effect of anoxia, lack of substrate and absence of metabolic waste removal. The tissues use stored glycogen and the leftover oxygen for continuous aerobic metabolism. However, this process soon stops due to depleted oxygen supply. In an effort to maintain tissue energy supply, glycolysis takes over, but this is not an efficient way to produce energy and the process also results in intracellular acidosis. While the pathophysiology of ischemia is complex,⁴ some of these events have been clearly delineated, such as depletion of high-energy phosphate, inhibition of cellular energy-dependent processes, generation of oxygen-free radicals, loss of osmotic balance across the membrane, stimulation of glycolysis, accumulation of metabolic products, intracellular acidosis and release of lysosomal enzymes.^{5 , 6} Short episodes of ischemia cause mild damage from which a cell can recover, while longer periods of ischemia cause irreversible cell damage, leading to cell death. An organ's tolerance to ischemia depends on the following three factors: (1) the size of the available stored energy pool; (2) the amount of the energy-demand per time unit; and (3) the efficiency of the anaerobically obtained energy.⁷ At 37°C, the maximum tolerable ischemic time is about 30 to 45 minutes for the heart,^{8 , 9} 30 to 60 minutes for the liver^{10 , 11} and less than 5 minutes for the brain.^{12 , 13} Beyond these time limits, irreversible damage results. Even if blood circulation is reestablished, total functional recovery is impossible.^{12 , 14} Furthermore, loss of function always precedes cell death. Some ischemic myocardial tissue, for example, can survive for 30 to 60 minutes, but cardiac muscle contractility is lost within less than 1 minute. Similarly, although neurons survive for some minutes, consciousness is lost almost immediately after a sudden interruption of blood supply to the brain.¹⁵

Metabolic Inhibition by Hypothermia

The development of hypothermia is based on the principle that reduced temperature decreases metabolism and oxygen consumption, so that organs can survive longer without nutrient supplements.⁸⁴ Cooling can hinder the rapid deterioration that occurs in an organ at 37°C when deprived of its blood supply. The precise mechanism by which hypothermia protects the viability of organs is very complex and not entirely understood. It is well known that hypothermia changes the reaction rate of all biochemical processes, especially the enzymatic reaction. This temperature dependence of reaction rates can be expressed by the Arrhenius equation:

Rate = Ae^-E/RT

where A is a constant known as the pre-exponential factor, T is the absolute temperature, R is the gas constant (8.31 J/mol/K), E is the activation energy (in J/mol) and e is a mathematical number of 2.71828.

An alternative method, the van't Hoff coefficient (Q₁₀), is derived from the Arrhenius equation. It is the decrease in the rate of reaction expressed as a percentage of the initial rate for a 10°C decrease in temperature. For every 10°C drop in temperature, the rate of a chemical reaction is decreased by approximately 50%. The decrease in the metabolic rate during hypothermia varies for the different metabolic processes, depending on the activation energy of the individual reaction, which can give Q₁₀ values ranging from 1.5 to 4. The Q₁₀ values for enzyme-catalyzed reactions usually fall between 1.5 to 2.5, whereas those for non-enzymatic reactions are usually in the range of 2.0 to 4.0.^{85 , 86} Experimental findings have indicated that the above estimations are suitable for many tissues and organs. Direct measurements of oxygen consumption by dogs showed a 50% reduction of oxygen consumption at 28°C and a 75% reduction at 20°C.⁸⁷ The mean value of oxygen consumption for the kidney is 6.26 ± 2.49 ml/min/100 g kidney weight at 39°C. At 30°C oxygen consumption is about 43% of the value at 39°C; at 20°C, it is only 16% of the control rate; and at 5°C, it is less than 5% (Fig. 1).^{85 , 88} However, hypothermia does not produce uniform inhibition of the cellular processes and the inhibition can vary by as much as 400% from one metabolic process to another after a decrease in temperature from 37°C to 0°C.⁸⁶ Bretschneider⁸⁹ suggested the following points of orientation for pure ischemia and for myocardial protection with nonbuffered solutions:

Figure 1

Oxygen consumption of rat tissues at different temperatures. Reprinted with permission from Fuhrman FA, In: Dripps RD, Ed. The Physiology of Induced Hypothermia. Washington, D.C. National Academy of Sciences, 1956: 50-51.

Q₁₀ for 35° to 25°C = 2.2

Q₁₀ for 25° to 15°C = 1.9

Q₁₀ for 15° to 5°C = 1.6

Q₁₀ for 35° to 5°C = 7

Some biochemical processes, especially those localized to cell membranes, show a step change in reaction rates at certain critical temperatures. These changes have been termed phase transitions and are thought to be the result of a change in the consistency of the cell membrane from fluid to gel. In mammalian tissues, phase transitions often occur at about 25°C to 28°C and may cause disturbed cell homeostasis. Biophysical processes, such as osmosis and water diffusion, are affected by temperature to a lesser extent, resulting in a linear change of about 3% per 10°C reduction in temperature.^{90 , 91} However, osmolar effects become pronounced as the freezing point of water is approached. Ice formation in tissue concentrates solutes in the residual nonfrozen cytosol, causing marked fluid shifts and membrane disruption. For this reason, the freezing point of water is the physical limit to the beneficial effects of hypothermia, unless protective chemical substances are present. Mammalian tissue will not regain function upon thawing from a frozen state, with the exception of some very simple systems, such as red blood cells.⁹²

The use of hypothermia in various medical applications began centuries ago.⁸⁷ The protection of hypothermia alone was demonstrated long before various preservation solutions were developed. Hypothermia and freezing techniques were used to store arteries for 1 to 35 days for transplantation early in the 20th century.^{93 - 95} From the 1920s to the 1940s, kidneys were transplanted experimentally after several hours or even within days of simple hypothermia preservation, but without long-term survival.^{96 - 98} Observations in the early 1950s confirmed that hypothermia significantly reduced heart and renal ischemic damage,^{99 , 100} and considerable effort was directed toward total protection of organ function with various types of regional hypothermia. In the late 1950s and early 1960s, it was established that reducing the metabolism of the kidney by cooling enabled it to withstand relatively prolonged periods of ischemia and long-term survival of experimental kidney transplants was reported after 4 to 24 hours of simple cold immersion or refrigeration.^{101 - 103} The simplicity of hypothermic storage stimulated researchers to use this technique as an alternative to perfusion and simple cold storage quickly gained popularity in the 1960s for the preservation of the heart,^{104 - 106} lungs^{107 , 108} and other organs.^{92 , 109}

Inhibition of Tissue Metabolism by Hibernation

Metabolic rate depression is an important survival strategy for many animal species and a common element of hibernation, torpor, aestivation, anaerobiosis, diapause and anhydrobiosis.^{129 - 132} Hibernation in mammals is a unique circannual adaptation allowing certain species, such as the ground squirrel, woodchuck, brown cave bat, European hedgehog and black bear, to survive extended periods of food deprivation when ambient temperatures may be well- below freezing. For example, hibernating ground squirrels conserve up to 90% of the energy that would be required if they remain active during the winter.¹³³ Moreover, it has also been suggested that hibernating animals age at a slower rate than those of the same species that are prevented from undergoing hibernation.¹³⁴ Profound metabolic changes accompany hibernation, including respiratory depression, a decline in body temperature and a cessation of feeding and renal function. These changes may be of great survival benefit to animals that can subsist without food and water for up to 5 months (up to 8 months in the arctic ground squirrel). In most winter hibernators, except the black bear, body temperatures decline to 4° to 6°C and even 1° or 2°C below freezing in the arctic ground squirrel.¹³⁵ It is notable that low temperature is not always required to save energy in some animals: hibernation also occurs in summer (aestivation), during periods of excessive drought and heat. Cactus mice, snails, Texas tortoise and some fish and crabs can hibernate in the summer time.^{136 - 138} These animals can save energy at very high temperatures. However, we know too little about the mechanisms related to aestivation.

Blood or its components from winter hibernating animals have been shown in studies to induce behavioral and physiological depression, including hypothermia, bradycardia, long-term hypophagia without significant weight loss, an anesthetized state and decreased renal function.^{139 , 140} Kidneys from hibernating animals were kept viable for up to 10 days.¹⁴¹ Studies have also shown that the erythrocytes in hibernating animals have an increased resistance to hemolysis and higher levels of unsaturated fatty acids in the cold blood taken during hibernation.¹⁴² Neither human cells nor ground squirrel cells would agglutinate in ground squirrel serum at low temperatures. During hibernation, stored fat becomes the primary metabolic fuel. Carbohydrate needs are met by gluconeogenesis from amino acids. Urea, the primary nitrogen-containing waste product of protein catabolism, is recycled rather than excreted, thereby negating the need for urination and hence arousal.^{143 , 144} Actively dividing cells, such as those of the intestinal epithelium, become relatively quiescent.¹⁴⁵

Despite the profound metabolic, biochemical and cellular changes noted in hibernating animals, relatively little information is available concerning the mechanism(s) that induce and maintain these changes or those involved in reversing them. One major reason for this problem is that most hibernation studies rely on in vivo systems, making experiential manipulation both difficult and expensive. For isolated organ preservation, it may not be necessary to induce whole- body hibernation. In the past, we have attempted to use the hibernation principle for the extension of isolated organs with some success.^{146 - 149} Subsequent experiments have also indicated protective effects using similar approaches.^{150 , 151} Plasma from hibernating animals and opioid agonists have both been used to induce a hibernation-like status in nonhibernating animals, but so far no single chemical or chemical combination has been able to induce true hibernation in either whole animals or in an organ. Thus, the theoretical advantages of inducing hibernation in organ preservation have not been successfully translated into the practice. Further characterization of the factors that induce both winter and summer hibernation may prove to be valuable toward extending organ preservation.

Metabolic Substrate Supplement

Supplementation of metabolic substrates to enhance high-energy phosphates represents a more active approach and efforts in this area have been ongoing for decades. The following are several major chemical groups that have shown protective effects in various scenarios. Our research group has focused on the use of fructose-1,6-bisphosphate (FBP, or fructose-1,6-diphosphate, FDP) and direct intracellular delivery of ATP. A more detailed report will be presented here.

Fructose-1,6-Bisphosphate (FBP)

Of all the glycolytic intermediates, fructose- 1,6-bisphosphate (FBP) has received substantial attention for organ preservation and other ischemic conditions. The glycolytic process (Embden-Meyerhof pathway) can be divided into two important phases. In the first phase, glucose is converted to FBP. Two of the enzymes catalyzing the reactions in this phase, hexokinase and phosphofructokinase (PFK), consume 2 ATP molecules. In the second phase, two molecules of glyceraldehyde 3-phosphate are ultimately metabolized to pyruvate and lactate with the production of 4 ATPs per glucose. Thus, the net production of one glucose molecule is 2 ATPs from glycolysis. If FBP were to enter the cell and be used as a substitute for glucose, it enters the glycolytic pathway beyond the two ATP-consuming steps and the two ATP molecules are saved.²⁰⁸ This results in double the ATP production in glycolysis. FBP has been used in various ischemic conditions, including hypothermic preservation of the liver,²⁰⁹ intestine,²¹⁰ skin flap,²¹¹ isolated heart perfusion,^{212 , 213} neuroprotection,²¹⁴ circulatory arrest²¹⁵ and sepsis.²¹⁶ Increased ATP production has also been reported by many investigators after using FBP.^{217 , 218}

The conventional view portrayed in biochemistry textbooks is that phosphorylated sugars such as FBP cannot cross the cell membrane. Although there have been some studies suggesting that FBP can be metabolized,^{218 , 219} there is no direct evidence that FBP can actually cross the cell membrane.

Our laboratory has studied the potential protective effect of FBP in cardiomyocytes and isolated heart preservation and beneficial effects were observed.^{220 - 222} We were especially interested in its possibility of enhancing glycolytic energy production. The following study was carried out to determine, in the absence of glucose, whether FBP could improve myocardial high-energy phosphate metabolism during hypothermic heart preservation. Our results indicated that adding FBP to St. Thomas solution did, in fact, improve high-energy phosphate reserves during hypothermic rabbit heart preservation.

Forty-two adult New Zealand White rabbits were used. Ten were used for normal controls (normal group), in which the hearts were biopsied fresh (without preservation) to obtain normal values of high-energy phosphate. Thirty- two rabbits were divided into two preservation groups for high-energy phosphate study. The animals were anesthetized with an intramuscular injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and chlorpromazine (0.5 mg/kg). The hearts were removed and preserved in cooled St. Thomas solution. In the study group (n = 15), FBP (5 mM) was added to St. Thomas solution. In the control group (n = 17), fructose (5 mM) was added instead. The hearts were preserved at approximately 4°C in a temperature-controlled refrigerator (Model 252, Sanyo Electric Co., Japan) for 18 hours.

After preservation, myocardial samples were taken from the left ventricle (LV), right ventricle (RV), left atrium (LA) and right atrium (RA) separately and high-energy phosphate and its metabolites were measured using HPLC on a Waters 2690 HPLC system with a Waters 996 photodiode array detector and Waters Millenium 32 manager software.

After 18 hours of hypothermic preservation, LV myocardial ATP concentrations declined in both the study and control groups as compared with normal hearts. The LV ATP content in the study group (1.45 ± 0.11 μmol/g wet weight) was about one third of the LV ATP in the normal group (4.42 ± 0.36 μmol/g WW, p < 0.05). However, LV ATP content in the control group (0.65 ± 0.12 μmol/g WW) was only 14% of the LV ATP in the normal group (p < 0.05, Fig. 2). ATP contents in the RV had a similar pattern (p < 0.05 study vs control). ATP contents in the atria also had similar patterns, but the difference between the study and control groups was not as statistically significant as in the ventricles. ADP content was also decreased, but the reduction was not as dramatic as the ATP. LV ADP content was still about 80% of normal in the study group, whereas it was only about 54% of normal in the control group (p < 0.05, study vs control, Fig. 3). ADP content in the RV had a similar change. AMP increased in the hearts after 18-hour preservation. In the control group, AMP levels in the LV increased to 156% of normal. It was also higher than that found in the study group (p < 0.05, Fig. 4). AMP concentration in the RV of the control group was nearly twice as high as that in the normal and study groups (p < 0.05). Total energy (TE = ATP + ADP + AMP) decreased in both preservation groups. In the study group, total energy (TE) in the LV was 58% of that in the normal group, but was still higher than that in the control group, despite the fact that the control group had a very high AMP level (p < 0.05, Fig. 5). The change in energy charge [EC = ( A T P + 1/2ADP)/(ATP + ADP + AMP)] exhibited similar patterns as that of ATP. The energy charge (EC) in the LV (0.58 ± 0.03) and RV (0.65 ± 0.03) in the study group was higher than those in the control group (0.37 ± 0.04 for LV and 0.43 ± 0.03 for RV, p < 0.05, Fig. 6). The ratio of ATP/ADP in the study group was 40% of that in the normal group, but the ATP/ADP ratio in the control group was about 25% of that in the normal group (p < 0.05).

Figure 2

Tissue adenosine triphosphate (ATP) contents after 18-hour preservation in the study (FBP) and control (fructose) groups compared with the normal hearts. In the study group, approximately 33% of normal ATP remained after preservation in each heart chamber, (more...)

Figure 3

Comparison of tissue adenosine diphosphate (ADP) contents in the study (FBP) and control (fructose) groups compared with normal hearts. There was approximately 80% of normal ADP content in the ventricle in the study group and approximately 60% of normal (more...)

Figure 4

Comparison of tissue adenosine monophosphate (AMP) contents in heart chambers among the 3 groups. Note the higher tissue contents of AMP in the ventricle in the control (fructose) group.

Figure 5

Profile of total energy in the study (FBP) and control (fructose) groups compared with the normal hearts. About 50% of total energy is present in the study group.

Figure 6

Profile of energy charge among the 3 groups. Approximately 80% of energy charge is still present in the study (FBP) group.

When the profile of high-energy phosphates was studied, adenine nucleotides in the four heart chambers in the study group maintained a similar pattern as in the normal hearts. ATP contents increased stepwise from RA, LA, RV, to LV. ATP was higher than ADP, which, in turn, was higher than AMP. However, in the control group, the high-energy phosphate profile was different: the content of ATP was similar in all chambers, but the ratio of ATP:ADP:AMP was reversed (Fig 7).

Figure 7

Comparison of adenine nucleotide profile among the 3 groups. After 18-hour preservation, the profile of adenine nucleotides in each heart chamber in the study (FBP) group is similar to that in normal hearts, but this profile is different or even reversed (more...)

In both preservation groups, the trivial amount of adenosine, xanthine and uric acid was nearly immeasurable in our HPLC analyses. However, IMP, inosine and hypoxanthine levels were increased in all four heart chambers. This increase was especially prominent in the control group.

This study clearly indicated that adding FBP to St. Thomas solution attenuated the decrease of high-energy phosphate reserves during hypothermic rabbit heart preservation.

Our results showed that after 18 hours of preservation, the LV still maintained about 30% of normal ATP levels and about 80% of normal EC in hearts treated with FBP. What do these values tell us? Although no causal relationship has been demonstrated, studies of myocardial ischemia have shown that when there is 80% depletion of myocardial ATP during the ischemic period, irreversible myocardial injury does not occur and adequate reperfusion should result in the eventual return of normal structure and function.^{55 , 223} When ATP levels are depleted more than 80% (between 20% and 10% of control), disturbances in cellular biochemical function and homeostatic mechanisms occur. Greater than 90% depletion of ATP levels (below 10% of control) is strongly correlated with irreversible myocardial injury.^{55 , 224} In vivo and in vitro heart studies have also indicated that an EC above 0.60 is generally associated with reversible myocardial injury, but an EC below 0.30 is associated with irreversible injury.⁵⁵ If these assumptions hold true in rabbit hearts, it may imply that when FBP is added for hypothermic rabbit heart preservation for 18 hours under hypothermia, although stunned, the myocardium may recover after proper reperfusion. This assumption is overly simplified, however. Long-term ischemia studies have shown that ATP levels continue to fall during the first 4 hours of reperfusion after global ischemia and a complete recovery may take as long as 10 days.¹⁸⁹

FBP is believed to have multifaceted beneficial effects, including augmentation of high-energy phosphate production during hypoxia,^{225 , 226} prevention of ischemia/reperfusion-induced leukocyte adherence,²²⁷ reduction of lipid peroxidation and oxygen free radical production,²²⁸ stimulation of nitric oxide synthase,²²⁹ increased red blood cell deformability and decreased blood viscosity,²³⁰ chelation of extracellular calcium²³¹ and prevention of platelet activation.²³² Many of the proposed beneficial effects of FBP can only be achieved in vivo, in the presence of blood, or during re-oxygenation. Our results were obtained in isolated hearts without the presence of blood or reperfusion. Thus, many of these effects can be excluded from consideration in our rabbit study. Augmentation of glycolytic energy production by FBP has been the most elusive and potentially significant mechanism; it remains the most logical explanation for our results.

Although the true mechanism by which FBP enhances glycolytic energy production is not clear, at least four possibilities have been proposed: (1) direct participation of FBP in glycolysis,²¹⁸ (2) indirect participation in glycolysis,²³³ (3) enhancement of glycolysis by activating glycolytic enzymes directly²³⁴ and (4) activation of glycolytic enzymes indirectly.²³⁵ Except for the first mechanism, the three other possible mechanisms can occur only in a solution containing glucose such as Euro-Collins solution or GIK solution. The St. Thomas solution used in this experiment did not contain glucose and any residual extracellular glucose would have been consumed shortly after the heart was removed. The enhancement of glycolysis induced by FBP would have to occur by the direct participation of FBP in glycolysis and this would require FBP to cross the cell membrane. However, the conventional view is that phosphorylated sugars, such as FBP, do not cross cellular membranes easily. In the past, a few studies have examined FBP movement through the cell membrane, but these studies provided little direct evidence to support this hypothesis. Results from our laboratories have provided some direct evidence that FBP does cross cell membrane.^{236 , 237} Several possible mechanisms have been proposed recently to explain this special feature of FBP. One mechanism is that FBP affects membrane stability, which allows normally nonpermeant FBP to passively diffuse across the membrane bilayer.^{236 - 238} This possibility was not explored in the past, but results from our laboratories have indicated that FBP can passively diffuse through artificial membrane bilayers and cellular membranes.²³⁶ Although the amount of FBP that can passively move through the membrane bilayers is small, this amount is still higher than normal FBP in the cytosol. Since the concentration of free FBP in the cytosol is only 1.2 μmol/L, an infusion of FBP in the millimolar concentration range results in a more than 1000-fold gradient across the plasma membrane.²¹⁹ Another mechanism is that FBP might be transported by a dicarboxylate transport system, because its utilization continues to increase at the highest concentration and its transport is inhibited by the dicarboxylate fumarate.²³⁹ Our more recent studies have also indicated that in isolated cardiomyocytes at 21°C, FBP uptake exceeded the uptake of L-glucose by several folds. This movement appeared to be via at least two distinct protein-dependent processes.²²² Because of its stability, ease of use and low cost, FBP may be a valuable metabolic intermediate for use as an adjacent chemical to improve organ function. More research should clarify some of the possible mechanisms.

Direct Supplement of ATP

Providing high-energy phosphate directly to the ischemic tissue is certainly an attractive idea. In fact, this has been an active project for more than a half- century. Direct infusion of free ATP or other high- energy phosphates would be a simple solution if it could be done. ATP entry into muscle cells was suggested in the 1944 study of Buchthal et al,²⁴⁰ but later work showed that ATP did not enter these cells.²⁴¹ Because the idea is so attractive, this finding did not discourage others from trying again. Indeed, administration of either free ATP or CP has been reported to improve tissue protection during ischemia in various models and species, such as in the heart,²⁴² kidney,²⁴³ liver,²⁴⁴ brain²⁴⁵ and also in various forms of shock.^{246 , 247} The theory is that since a significant amount of ATP is released into the extracellular space by several types of cells (myocytes, cardiomyocytes, brain cells), ATP can cross cell membranes.^{248 , 249}

However, this theory has not gained wide acceptance in the scientific community; experimental and clinical studies have not duplicated the many promising results. As described below, strongly charged molecules like ATP normally cannot pass the cell membrane without a transporting mechanism.^{242 , 250} Besides, the half-life of ATP in the blood is less than 40 seconds, which poses technical difficulties for maintaining the ATP supply.²⁵¹

All cells are surrounded by a plasma membrane that is composed of lipids, proteins and carbohydrates. The basic structure of the plasma membrane is the phospholipid bilayer, which is impermeable to most water-soluble molecules and is responsible for the basic function of membranes as barriers between two aqueous compartments. The plasma membranes of animal cells contain four major phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin). These phospholipids are asymmetrically distributed between the halves of the membrane bilayer. The internal composition of the cell is maintained because the plasma membrane is selectively permeable to small molecules. Larger, especially charged molecules, for which no specific transport mechanisms exist, cannot cross cell membrane under normal conditions. Specific transport proteins (carrier proteins and channel proteins) are needed for the selective passage of these molecules across the membrane (Fig. 8). Unfortunately, ATP and many energy-rich glycolytic intermediates belong to this category.²⁵² Although the presence of equimolar MgCl₂ reduces the negative charge of ATP from 3 to 1, this does not alter the permeability of ATP because no known transporting mechanism exists in the cell membrane. Results from our laboratory have indicated that a small amount of free ATP can be taken up by the cells, most likely through some pores (about 0.8 nm in diameter) in the cell membrane.⁶⁵ The question is whether this small amount can support tissue metabolism.

Figure 8

Permeability of the cell membrane phospholipid bilayers. Gases, hydrophobic molecules and small polar uncharged molecules can diffuse through phospholipid bilayers. Larger polar molecules and charged molecules such as ATP cannot. Reprinted with permission (more...)

For the past half century, using various carriers to deliver impermeant substances into the cytoplasm of cells has been a major research interest for cell biologists and clinicians. The ideal system for such delivery would couple both specificity and the ability to easily target multiple cells. Five primary approaches have been used to bypass the plasma membrane barrier: permeabilization, microinjection, polymer delivery, liposomal encapsulation and microemulsion. Permeabilization, which allows entry of materials found in the external medium, has been attained by electroporation and by pore-forming proteins or detergents.^{253 , 254} However, the lack of specificity and difficulty in controlling this technique limit its application predominantly to research. Furthermore, as soon as nonspecific pores are formed in the cell membranes by permeabilization, many unwanted molecules can also food in, loading the cells with toxic chemicals and losing important membrane ion gradients. While micro-injection allows a more targeted delivery than the permeabilization technique,^{255 , 256} this is a time-consuming and technically challenging procedure. This procedure, therefore, has limited clinical application. The recent development of biodegradable polymers may offer a new approach for this purpose,^{257 , 258} but at this time, the main interest is focused on the sustained release of some drugs because polymers are too large to penetrate the cell membrane.^{259 , 260} Microemulsion can produce much smaller oil droplets, making efficient oil-in-water delivery of drugs.^{261 , 262} However, ATP is highly hydrophilic and does not dissolve in oil.

One promising approach is the loading of medication into liposomes, or multilamellar vesicles, which are microscopic sacs made of the very phospholipids that constitute cell membranes. When a high enough concentration of phospholipids is mixed with water, the hydrophobic tails spontaneously herd together to exclude water. In contrast, the hydrophilic heads bind to water. The result is a bilayer in which the fatty acid tails point into the membrane's interior and the polar head groups point outward facing the water. As a liposome forms, any water-soluble molecules that have been added to the water are incorporated into the aqueous spaces in the interior of the spheres, whereas any lipid-soluble molecules added to the solvent during vesicle formation are incorporated into the lipid bilayer.²⁶³ Liposomes can be filled with a variety of medications and, because of their similarity to cell membranes, are not toxic. They also protect their loads from being diluted or degraded in the blood. As a result, when the liposomes reach diseased tissues, they deliver concentrated doses of medication. Individual liposomes were shown to be capable of carrying tens of thousands of drug molecules, making them an efficient carrier for delivery of certain drugs.²⁶⁴ However, three major problems have limited the widespread use of liposomes for cytosol drug delivery: (1) liposomes are not readily fusogenic, mainly because the stored energy of the vesicles radius of curvature is minimal and the internal layers may inhibit fusion.^{265 , 266} (2) Because of their size, most intravenously infused liposomes are unable to leave the general circulation, except in areas where vessels become leaky (such as inflammation).²⁶⁴ (3) The body's immune system recognizes the liposomes and removes them from circulation, regardless of the vesicles' composition and size. The liver, spleen and bone marrow take up nearly all liposomes given intravenously, preventing liposomes from circulating long enough to reach many targeted cells and tissues effciently.²⁶⁴ To circumvent these problems, various techniques have been developed such as coating liposomes with polyethylene glycol,²⁶⁷ polyvinyl alcohol,²⁶⁸ or other polymers²⁶⁹ to make them “stealth” and combining liposomes with hemagglutinating virus of Japan (HVJ) to enhance fusion.²⁷⁰

Our laboratory has focused on specially formulated, highly fusogenic and very small unilamellar lipid vesicles (with diameters around 120-160 nm) to encapsulate Mg-ATP. Lipid vesicle fusion (and the delivery of ATP) occurs primarily by one of four methods: (1) increasing electrostatic interactions; (2) destabilizing the membrane bilayer; (3) increasing nonbilayer phases; or (4) creating dissimilar lipid phases. By changing the phospholipid composition through the use of one or all of these methods, a highly fusogenic lipid vesicle can be created: altering the charge of the phospholipid head group, increasing mean molecular area of the lipids, creating dissimilar regions of lipids and increasing the kinetic energy of the lipid vesicles. The fusogenicity of vesicles is strongly influenced by the dynamic and structural properties of the lipid membrane. The existence of a heterogeneous lipid-bilayer structure, which is composed of fluctuating gel and fluid domains that are prevailing in the temperature range of the main phase lipid membrane transition, leads to a strong increase in the fusion rate. Additionally, membrane curvature stress and the incorporation of various nonlamellar forming agents into the lipid membrane has a dramatic effect on the fusion properties.²⁷¹ Several formulations have been tested and the chemicals used to make ATP-vesicles included L-α-phosphatidylcholine (Soy PC), Mg-ATP, polyethylene glycol, chloroform and trehalose. Our unilamellar vesicles were found to be highly fusogenic. In our study of endothelial culture, it was clearly shown that these vesicles fused with the cells in 5-10 minutes, delivering water-soluble carboxyfuorescein (Fig. 9). In one rat study, primary cardiomyocytes were incubated in either (1) ATP-vesicles, (2) lipid vesicles only, (3) Mg-ATP (5 mM), or (4) culture medium. After equilibration, potassium cyanide (KCN, 5 mM) was added to the culture media for 30 minutes. Results indicated that cardiomyocytes after 30 minutes of chemical hypoxia had the highest ATP and TE contents in the group treated by ATP-vesicles. Neither vesicles alone nor ATP alone had a similar effect (p < 0.05, Fig. 10). The redox status of cardiomyocytes was decreased by the use of KCN. However, in the ATP-vesicles group, this decrease of redox status was significantly different from the other groups. The Alamar-Blue fluorescent intensity reading with ATP-vesicles was higher than with vesicles only, free Mg-ATP only, or with M199 (Fig. 11). Cardiomyocyte contractility was higher (Fig. 12).

Figure 9

Endothelial uptake of fluorescein-encapsulated small unilamellar lipid vesicles within 5-10 minutes.

Figure 10

Comparison of ATP and total energy phosphate (TEP) in cardiomyocytes under chemical hypoxia indicates ATP-vesicles increase ATP and TEP by ∼2 fold (p < 0.05). Rat cardiomyocytes were incubated with KCN (5 mM) for a period of 30 minutes. (more...)

Figure 11

Redox status of cardiomyocytes under chemical hypoxia is increased by ATP delivery. Rat cardiomyocytes were incubated in either media or media + KCN (5 mM). After 30 minutes, the redox status of the cells was determined using Alamar Blue. ATP-vesicles (more...)

Figure 12

Cardiomyocyte contractility is maintained under chemical hypoxia by ATP-vesicles. The cells were stimulated with 0.5-4 Hz, 8-volt electric stimulator (MyoPacer™) and contractile velocity and duration of contraction were recorded after a 30-minute (more...)

During the process, we also have resolved several problems that were expected when highly fusogenic lipid vesicles are used. One problem was the stability of these vesicles. Although it has been proven that small unilamellar lipid vesicles are more stable in blood circulation,²⁷² it is possible that these vesicles may fuse with each other when left alone for a long period of time.²⁷³ We have used a freeze-dry technique to keep these vesicles stable without particle size change during storage. Another problem was the temperature effect. All lipids have a phase transition temperature that will affect the fusion speed.^{271 , 274} Our current vesicles are created well above the liquid-crystalline-to-gel phase transition, circumventing the problem of lipid phase separation. Efforts are being made to further improve the delivery efficiency. Further research work may prove to be fruitful.

Other Pharmacologic Interventions

Drugs that can provide a physical environment to enhance the hypothermic effect or provide chemical protection, either directly or indirectly, have also been used in organ preservation with some success. So far, none of the drugs is dramatically effective in itself, but they have been reported as beneficial when used as adjuncts to enhance the quality of storage. Some of these drugs are listed below. Agents for specific organs, such as high potassium in cardioplegia and surfactant for lung preservation,²⁷⁵ will not be discussed here.

Preconditioning

The earliest study of preconditioning was probably performed more than 4 decades ago by Dahl and Balfour.³³⁷ They found that the ischemic survival of rats was extended by placing them in nitrogen for a short period of time, letting them recover and then inducing anoxia. They also found that concentrations of lactate, pyruvate and ATP in the brain were higher during anoxia in the group subjected to pre-anoxia. When glycolysis was inhibited by iodoacetate, the pre-exposed advantage disappeared. Dahl and Balfour believed that the preconditioning was related to a faster production of ATP during anoxia because the first anoxia resulted in higher pyruvate and lactate reserves.

The phenomenon of preconditioning was described in 1986 by Murry et al,³³⁸ who discovered the protective effect of a brief ischemic period with regard to the detrimental consequences of subsequent prolonged ischemia. Since then, preconditioning has been shown to limit infarct size and to reduce ventricular arrhythmias during sustained ischemia and reperfusion. Brief repetitive periods of ischemia have been shown to retard cardiac energy metabolism during sustained ischemia in various animal experiments. This inhibition leads to sparing of high-energy phosphates and improves the time-averaged energy state during ischemia.^{339 , 340}

Other Important Factors in Organ Preservation

Nature Still Provides the Best Preservation

While various attempts have been made to further extend cold preservation time for solid organs, it is disappointing to realize that decades of research studies have not extended preservation times for the heart and lungs, even for a few hours. It has been shown repeatedly and is also reflected in this book, that using perfusion can extend survival time. This is obvious because perfusion can supply oxygen and nutrients continuously and remove metabolic wastes—all are critical to organ survival. There are complicated issues regarding using the perfusion technique. For one thing, unlike single-flush hypothermic preservation that needs little attention during the preservation period, mechanical perfusion is labor intensive and expensive. It is difficult to gain acceptance in the managed care era. Perfusion itself can pose additional risks to the organs that are not present in hypothermic storage, such as damage to vascular endothelial cells, the possibility of mechanical failure and particle formation in the perfusates.^{92 , 350} To overcome these problems, a simple heart-lung autoperfusion was developed that did provide longer preservation time for the heart, but not the lungs.^{351 - 355} We tried this technique and found one possible reason for lung damage: the circulating aggregates from blood-air (and possible artificial circuit) interface.³⁵⁶ We eliminated the blood-air interface and expanded the organ block to include the heart, lungs, liver, pancreas, duodenum and both kidneys. In that preparation, the heart pumps blood, the lungs oxygenate the blood, the liver processes normal chemical reactions and metabolic wastes are removed by the kidneys. The organs are self-contained and a respirator is the only artificial equipment needed for its survival. The organs could be preserved for more than 2 days with good function.^{146 , 147 , 357} In one study, using 6 pairs of adult mongrel dogs, the organs were preserved from 24 to 33 hours (mean 26.7 ± 1.4 hours) in the multiple organ block along with intermittent infusion of plasma from hibernating animals. Orthotopic lung transplantation was performed and the dogs were observed for 24 hours with good lung function.¹⁴⁹ Figure 13 is an example of the lungs at 30 hours of preservation. These lungs show the best quality of any preservation technique we have seen.

Figure 13

An example of the lungs at 30 hours of preservation. Not only do the lungs look normal, tissue wet/dry ratio was also normal.

Metabolic management during perfusion is not the main focus in this chapter because it requires totally different strategies. However, the above example indicates that physiologically, using an animal's own heart as a pump and its own blood as a perfusion medium, provides the simplest and possibly the best environment for long-term organ survival. The normothermic autoperfusion technique was used early in the history of organ preservation. Due to reasons similar to mechanical perfusion, it is probably difficult to gain acceptance in clinical practice. Because of the technical difficulties in the setup and the monitoring processes, even the simplest autoperfusion was used only sporadically.^{292 , 358 , 359} We do not know the other possible mechanisms of the plasma from hibernating animals in this preparation, except its obvious role in reducing liver congestion.^{146 , 147 , 357} The above example appears to show that using natural circulation is still the best option for long-term organ preservation.

Future Perspective

Over the past 40 years, slow progress has been made in organ preservation for transplantation. Despite the extension of preservation times for the kidneys and livers, safe preservation times for the heart and lungs are still very short.³⁶⁰ In the past decade, there has been a gradual expansion of marginal donor organs used as a result of a more careful selection of solutions for individual organs,^{361 , 362} but there has not been a dramatic extension in preservation times.³⁶³ Longer ischemic times are often associated with early heart failure and chronic fibrosis after transplantation.^{364 - 366} Many of the compounds mentioned in this chapter, albeit with great theoretical advantages and promising experimental results, have not made their way into clinical practice. This is mainly because sustained protective results are lacking in a larger number of animal experiments and/or clinical trials. Among the reasons for this is the period of organ ischemia outside the body, which is subject to a number of biochemical stress factors that cause a combined-damage effect that cannot be prevented or treated by a single drug. Another reason is that our knowledge of known critical components of tissue damage is still incomplete. For example, although the decline of energy production plays a major role in tissue ischemic damage and cell death is well recognized, the critical point at which cells die is far from settled. Evidence shows that it is very unlikely that there is a single critical level of ATP for all tissues and organs. Even in the same organ, this critical level may be different due to the existence of other factors. As such, it is not surprising to realize the controversy toward the relationship between high-energy phosphate concentration and organ function and viability. In the literature, both positive^{54 , 367 , 368} and negative^{72 , 81} relationships have been reported. There is also the possibility of ATP compartmentation in subcellular organelles. Our current techniques can only determine the total tissue content of a particular metabolite and may not provide a good estimate of its concentration in a specific subcellular compartment. If a large concentration gradient of any compound across the mitochondrial membrane is present, the interpretation will be difficult.³⁶⁹ Furthermore, it is not the sole concentration of ATP that is critical to cell survival, but it is the rate of ATP turnover that is more important to the survival of the cells.³⁷⁰ This will gradually change when our knowledge of biochemical and immunological mechanisms of organ damage improves.

Further improvement of preservation techniques is critical with the increased use of marginal and suboptimal donor organs and organs from nonheart-beating donors (NHBD). However, organ preservation is only one ring in the chain of organ damage, beginning before retrieval and ending at restoration of blood supply. Many of the organs from NHBD are severely damaged during the long period of hypotension before retrieval, making preservation more critical.

True long-term organ preservation for months or years would be a valuable future development. Such techniques would transform transplantation into elective surgery, probably eliminate recipient waiting lists, make the process of time-consuming matching of donors and recipients more possible and provide time for immunological pretreatment of the recipients or donor organs.³⁷¹ It is unlikely that the current hypothermic storage or mechanical perfusion techniques will achieve this goal. However, hypothermic storage is simple and the most cost-effective. A gradual extension of current hypothermic preservation time will be possible when there is more understanding of the specific metabolic demands and physiologic requirements of different cells and organs. This would include the nature of hypoxic damage to cells and organs and the methods that avoid or reduce various kinds of damage. It may require a combined treatment of several drug categories to offset injuries caused by different mechanisms,^{60 , 372 , 373} and the treatment is probably organ-specific.³⁷⁴

Acknowledgements

This study was supported in part by NIH grants HL64186, DK74566 and AR52984. The author wishes to thank Drs. Dongping Hua, Benjamin Chiang and William Ehringer for their experimental contributions; Jiusheng Ye for his HPLC work and Margaret A. Abby for her editorial assistance.

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