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JOURNAL OF NEUROTRAUMA Volume 9, Number 2, 1992 Mary Ann Lieben, Inc., Publishers

Brain Levels of Polyethylene Glycol-Conjugated Superoxide Dismutase Following Fluid Percussion Brain Injury in Rats KENSHI

YOSHIDA,1

GREGORY F. EARL F.

BURTON,2 HAROLD YOUNG,1 ELLIS3

and

ABSTRACT

Polyethylene glycol-conjugated Superoxide dismutase (PEG-SOD) is being explored as an agent to reduce oxygen radical-mediated damage following brain injury. Yet little is known concerning the site of action of IV-administered PEG-SOD or the capacity of this conjugated enzyme to enter the brain. The purpose of this study was to determine the brain content of PEG-SOD in normal and fluid percussion injured rats. The fluid percussion device was attached over the right parietal cortex and a moderate (2.0 atm) intensity injury was produced. PEG-SOD was conjugated with 125I and given (2000 U/kg, 5 u-Ci/kg) to rats either 30 min before or 30 min after brain injury. Another group received [l25I]PEG-SOD but was not injured. Plasma and left and right brain hemispheres were counted for [125I]PEG-SOD. Plasma levels of [125I]PEG-SOD declined similarly in all three groups during the 90-min period after IV administration. Brain [l25I]PEG-SOD was low in control animals (0.034 U/g wet wt). In animals given PEG-SOD after injury the brain level was elevated sixfold in both the left and right hemispheres, compared to control. In rats given the drug before injury, [I25I]PEG-SOD was 10 times control level in the right hemisphere, which is the side on which the injury device is attached, and 6 times control level in the left hemisphere. We conclude that traumatic brain injury produces an increase in brain PEG-SOD. The exact cellular site of the increased brain PEG-SOD remains to be clarified.

INTRODUCTION biochemical factors contributing to the sequelae of traumatic injury to the CNS are complex. Several laboratories have provided evidence that oxygen radicals may be important contributors to the generation of posttraumatic abnormalities of the microcirculation or tissue edema (Kontos and Povlishock, 1986; Kontos and Wei, 1986; Means and Anderson, 1987; Faden, 1987; Hall and Braughler, 1988). The protective effects of enzymes, such as Superoxide dismutase (SOD) and catalase, have been well documented, thus linking the scavenging of oxygen free radicals with cytoprotection (Wei et al., 1981; Kontos and Wei,

The

'Department of Surgery, Division of Neurosurgery, department of Microbiology and Immunology, 3Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia. 85

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YOSHIDA ET AL.

1986; Kontos, 1989). Superoxide dismutase, when administered as a constant infusion, has been shown to prevent the increase in cerebrovascular permeability and brain edema normally caused by acute hypertension (Zhang and Ellis, 1991). A distinct disadvantage to native SOD is its short circulating half-life (McCord and Fridovich, 1969;Turrenset al., 1984; Odlindetal., 1988). To overcome this drawback, several investigators have conjugated SOD to polyethylene glycol (PEG), thus decreasing SOD's rate of metabolism and resulting in a half-life of approximately 40 h (Beckman et al., 1988). PEG conjugation has been reported also to facilitate uptake of SOD into cultured endothelial cells (Beckman et al., 1988). Previous studies have shown that SOD has a plasma half-life of approximately 6 min, has low entry into the brain, and is quickly taken up into the kidney and urine (Odlind et al., 1988). The distribution of PEG-SOD into brain and other tissues has not been well studied. We recently used [ l25I]PEG-SOD to study distribution

and found that minimal amounts of [i25I]PEG-SOD become associated with the normal rat brain and that PEG-SOD reaches the highest concentration in the kidney. We also found that acute hypertension more than doubles brain-associated PEG-SOD, but only when PEG-SOD is given before hypertension. The purpose of the present study was to determine if a more extensive injury, produced by fluid percussion experimental brain injury, will increase brain content of l25I-labeled PEG-SOD. The relevance of these studies is underscored by the fact that PEG-SOD currently is being given to brain-injured humans in a clinical trial.

MATERIALS AND METHODS Iodination

of PEG-SOD

Iodination of PEG-SOD (Sigma Chemical Co., St. Louis, MO) was performed using a modification of the procedure of Beckman et al. (1988). Briefly, 15 p.g of PEG-SOD (in 100 p.1 Dulbecco's phosphate-buffered saline) was mixed with 2 mCi of 125I (Amersham, Arlington Heights, IL) and added to a glass culture tube previously coated with l,3,4,6-tetrachloro-3a,6a-diphenylglycouril (Iodogen, Pierce Chemical Co., Rockford, IL). The reaction mixture was incubated at room temperature for 20 min, and the reaction was stopped by removing the liquid phase. Unbound iodine was removed by adding the iodine-protein mixture to an Eppendorf tube containing G-25 (Pharmacia, Piscataway, NJ), centrifuging for 30 s at 900g, and collecting the supernatant. The PEG-SOD was labeled to a specific activity of 21-28 u.Ci/u.g protein. The iodinated PEG-SOD retained 78% of its Superoxide scavenging capacity, as determined by the cytochrome C reduction assay for Superoxide (Capro et al., 1978). The material was stored at 4°C until used.

Animal

Preparation

and

Injury Protocol

Twenty 300-400 g male Sprague-Dawley rats were used. After anesthesia was induced with IP pentobarbital (50 mg/kg), the animals were tracheostomized and ventilated with room air. The ventilatory rate and volume were adjusted so that the end-expiratory C02 was maintained at approximately 30 mm Hg. A femoral artery and vein were cannulated for blood sampling and IV infusion. Blood gases and pH were analyzed to insure adequate and consistent ventilation. The temperature of all animals was maintained at 37°C with a heating pad. The method for producing fluid percussion brain injury has been published in detail previously (Dixon et al., 1987). The protocols were approved by our institutional Animal Care and Use Committee. Briefly, a craniectomy was made over the right parietal area, and the dura was left intact. An injury attachment device was implanted into the craniectomy and held in place with dental acrylate. The fluid percussion device consists of a fluid-filled cylinder that has a movable piston on the end. The device is attached to the animal such that the cranial vault is continuous with the fluid-filled cylinder. A weighted pendulum is dropped and strikes the piston, thus producing a transient pressure pulse that enters the cranial vault and produces injury throughout the cranial vault. The pressure pulse wave is 20 ms long and is highly reproducible. The pressure injury used in these experiments was 2.0 atm and, thus, moderate in intensity. The duration and intensity of the fluid percussion pressure wave were recorded on a storage oscilloscope. 86

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BRAIN SOD AFTER INJURY

[ l25I]PEG-SOD (5 (xCi/kg) plus unlabeled PEG-SOD (2000 U/kg) was given in 0.2 ml of saline through the femoral vein. The total amount of protein administered was less than 1 mg. Plasma samples (0.1 ml) were collected through the arterial line at 1, 5, 30, 60, and 90 min after PEG-SOD infusion. All animals were killed at 90 min after administration of PEG-SOD. The animals were divided into three experimental groups (Fig. 1 ). One group received only [ l25I]PEG-SOD and was not injured. Another received [ l25I]PEG-SOD 30 min after fluid percussion injury, and a third group received [ ,25I]PEG-SOD 30 min before fluid percussion injury. At 90 min after injection of PEG-SOD, a CSF sample was collected by cisternal puncture in the uninjured group only. After the rats were killed, they were perfused with saline via the left ventricle at a pressure of 100 mm Hg in order to remove intravascular f'25I]PEG-SOD. Therefore, all radioactive counts reported are in the vascular wall or are extravascular. The brain was removed and divided into left and right hemispheres. Each hemisphere was weighed, and then the l25I was counted with a gamma counter. The permeability of the blood-brain barrier or the blood-CSF barrier to [l25I]PEG-SOD was calculated by dividing the cpm per gram of wet brain by the cpm per milliliter of plasma and then multiplying by 103. The exogenous PEG-SOD concentration in each sample was estimated from the radioactive counts in the tissue by use of the specific activity of the total [l25I]PEG-SOD infused. Results were examined by analysis of variance followed by the Scheffe F-test. The data are expressed as mean ± SEM. A p value -

(120 min post TBI)

TBI Post-treatment

(TBH PEG-SOD)

-30

+90 +30 +60 Time of PEG-SOD circulation (min)

0

PEG-SOD

PEG-SOD

=

(2000 U/kg

+

5

uCi/kg, i.v.)

TBI

=

traumatic brain

injury

Experimental paradigm. [125I]PEG-SOD was given IV 30 min before or after experimental fluid percussioninduced traumatic brain injury (TBI). Animals were subjected to a moderate (2 atm) intensity injury. Animals were killed at 90 min after receiving [l25I]PEG-SOD. Control animals received [125I]PEG-SOD but were not injured.

FIG. 1.

87

YOSHIDA ET AL. Table 1.

Control Physiologic Parameters

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Groupa Control

Pretreatment

Posttreatment

(PEG-SOD, noTBIh) (n 5)

(PEG-SOD+TBI) (n 5)

(TBI+PEG-SOD) (n 6) =

=

=

Mean arterial blood pressure

;i2± 3

Control maximum Control blood gases and

Postinjury

113 ±4 172 ± 7

115 ± 7 176 ± 7

pH 85.2 ± 4.6 29.0 ± 0.4 7.42 ± 0.01

Pao2 (mm Hg) PaC02 (mm Hg) Blood pH

96.5 30.2 7.45

97.3 ± 2.9 29.6 ± 0.7 7.44 ± 0.01

±

5.4 0.5 ± 0.01 ±

significant differences were found in the three groups. bTBI, traumatic brain injury.

aNo

hemispheres in the postinjury treatment group, being 0.21 ± 0.047 and 0.21 ± 0.053 U/g wet weight, respectively. In contrast, the PEG-SOD concentration in the right hemisphere (0.35 ± 0.014 U/g wet weight) was significantly increased over the left hemisphere (0.20 ± 0.022 U/g wet weight) in the preinjury treatment group (Fig. 3). Permeability of the blood-cerebrospinal fluid barrier to [125I]PEG-SOD in the sham-operated control group was represented by a CSF/plasma [l25I]PEG-SOD ratio of 3.59 x 10~3. We were unable to reliably obtain CSF samples in injured rats. Table 2 shows changes in permeability of the blood-brain barrier to PEG-SOD in each hemisphere of each group. There was a significant increase in permeability of the blood-brain barrier to PEG-SOD in both hemispheres of both groups of injured animals. In animals pretreated with [l25I]PEGSOD, there was a larger increase in permeability of the right cortex, which is the side under the attachment of the fluid percussion device. Plasma PEG-SOD Concentration Following IV Administration 40

(PEG-SOD, no injury), n=5 pretreatment (PEG-SOD - TBI), n=5 posttreatment (TBI -* PEG-SOD), n=6 control

E a o 30 (oi

(5 UJ Q.

"

20

a

E

(0

«

O.

10

0

20

40

60

Time post PEG-SOD

80

100

(min)

FIG. 2. Plasma [,25I]PEG-SOD concentration following IV administration. Plasma levels decreased significantly during the first 30 min after injection and then decreased minimally between 30 and 90 min. Analysis of variance showed that there were no significant differences between groups at each individual time point. 88

BRAIN SOD AFTER INJURY 0.4

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m

S 0.3

**

Right hemisphere M Left hemisphere

i

|

3 a

O 0.2 A V) I

o

UJ Q.

8 0.1 ç

'2m

o.o n=5 control (PEG-SOD, no injury)

n=5

n=6

pretreatment

posttreatment

(PEG-SOD -> TBI)

(TBI -* PEG-SOD)

FIG. 3. [I25I]PEG-S0D concentration in brain. [ l25I]PEG-SOD concentration was determined 90 min after injection of the enzyme. There was a 6-fold elevation of the brain label in animals given SOD after brain injury and a 6- to 10-fold elevation in those given the radiolabeled enzyme before injury. *p < 0.05 compared to control. **p < 0.05 compared to all other groups.

DISCUSSION

Oxygen radical production has been implicated in the pathogenesis of a number of different kinds of brain injury (Kontos and Povlishock, 1986; Kontosand Wei, 1986; Means and Anderson, 1987; Faden, 1987; Chan etal., 1987, 1991; Hall and Braughler, 1988). As recently reviewed by Traystman et al. (1991), brain damage and abnormalities that occur following reperfusion of the ischémie brain can be prevented by treating systemically with radical scavengers, including PEG-SOD. However, a study by Haun et al. (1991) aimed at measuring increases in brain SOD activity associated with improvement in outcome failed to show an increase in brain-associated SOD after systemic administration of PEG-SOD. Interpretation of these findings is somewhat complex, although two possible explanations may be offered. First, the beneficial effect of SOD may be caused purely by elevation of intravascular SOD levels. A second possibility is that such a small tissue enhancement of SOD is necessary for therapeutic action that this increase could not be measured reliably by the cytochrome C reduction assay for SOD. As noted by Haun et al. (1991), this latter possibility may be especially likely if brain SOD enrichment is limited only to the vasculature. Table 2.

Brain/Plasma Ratios

of

i25I PEG-SOD

Ulg

wet wt brain U/ml plasma

Right hemisphere Left hemisphere

.

]q3 (-

±

Control

Pretreatment

Posttreatment

(PEG-SOD only) (n=5)

(PEG-SOD--TBI) (n 5)

(TBI+PEG-SOD) (n 6)

1.70 1.87

=

0.29 0.22

24.1 ± 2.8** 14.2 ± 3.0*

*p < 0.05 compared to control. **p < 0.05 compared to all other groups. 89

=

12.6 12.6

± ±

3.6* 3.6*

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YOSHIDA ET AL.

Systemically administered SOD has a very short plasma half-life. To circumvent this, we and other investigators have used polyethylene glycol-conjugated Superoxide dismutase, which has a long half-life of approximately 40 h. The PEG-SOD compound offers the potential therapeutic advantage of a single administration to the injured patient. Our current data in rats show that, after an initial decline, circulating plasma levels of PEG-SOD are relatively stable up to the 90 min of observation. In the current study, just as in our other recent studies with

[ 125I]PEG-SOD (Yoshida et al., 1992), we found

our current results show that when brain 30 min after fluid administered percussion injury a sixfold elevation of PEG-SOD [l25I]PEG-SOD occurred in both brain hemispheres. The increase in brain-associated PEG-SOD is, therefore, not likely a result of hypertension alone, since the PEG-SOD was administered approximately 30 min after the hypertensive insult that follows fluid percussion brain injury. The sixfold PEG-SOD increase in both hemispheres in fluid percussion-injured animals must, therefore, be due to some permanent or continuing mechanism that allows increased brain-associated [l25I]PEG-SOD. When [125I]PEG-SOD was given before trauma, there was a sixfold increase in SOD activity in the left hemisphere, which was similar to the SOD elevation in both hemispheres of rats given PEG-SOD after injury. Interestingly, the right hemisphere in rats given PEG-SOD before injury had a significantly greater level of PEG-SOD than the left hemisphere. The 10-fold increase in PEG-SOD in the right hemisphere suggests that trauma may be greater on the side of the brain under the attachment of the brain injury device. In fact, previous experiments examining posttraumatic permeability to protein and brain water content suggest that injury is greatest directly under the site of attachment of the brain injury device (Ellis et al., 1989). Increased SOD in the right hemisphere may be caused by several mechanisms. One of these might include a generalized increase in blood-brain barrier permeability. Alternatively, the increased level of PEG-SOD on the right side may be caused by hemorrhage. However, with this level of injury (2 atm), bleeding is not a predominant feature. Regardless of the cause of the increased permeability on the right side, comparison of the pretreatment and posttreatment groups shows that the mechanism allowing for the increased permeability on the right side is gone by 30 min after injury, since postinjury administration of SOD was associated with the same sixfold increase in SOD activity in both the left and right hemispheres. This transient greater increase in permeability suggests the likelihood of microbleeding associated with the traumatic event. As reviewed here, administration of SOD has been shown to reduce brain injury in several different animal models. Yet the exact site at which the SOD is acting to reduce brain injury is uncertain. In the current studies, we measured total hemispheric [125I]PEG-SOD. We have not been able to determine the exact cellular location of this increase in SOD activity. It may be that the normal or trauma-induced increase in SOD activity is vascularly associated. Indeed, previous studies have shown increased pinocytosis in the vascular endothelium after experimental fluid percussion brain injury (Povlishock et al., 1978). If we assume that all [l25I]PEG-SOD is associated with the vasculature, the local vascular concentration would be much higher than that shown in Figure 3. If, for example, we assume that the weight of the vascular wall is approximately 2% of total brain weight and that all SOD activity is in the vasculature, the concentration per unit tissue in the vasculature would be approximately 50 times the 0.2 unit per gram weight shown for injured brain in Figure 3, or approximately 10 U/g wet weight of vascular tissue. Our previous experiments have shown that 2000 U/kg has a positive effect in experimental animal studies (Zhang and Ellis, 1991). Recently emerging, yet preliminary data from a PEG-SOD trial in humans suggest that at the 10,000 U/kg dose, PEG-SOD may be decreasing death and the vegetative outcome. Extrapolating this theoretical discussion concluding that there may be a 10 U/g concentration in the vascular tissue associated with the 2000 U/kg dose and multiplying our 2000 U/kg dose by the factor of 5 to get 10,000 U/kg would produce a vascular tissue concentration of approximately 50 U/g. This concentration is near the 60 U/ml concentration that has been shown to be effective when applied topically in animal models of fluid percussion brain injury. Our discussion of potential vascular concentrations of PEG-SOD is, at the least, highly theoretical and speculative. It points out, however, that if administered PEG-SOD is concentrated in one brain cell type or space, it may reach much higher local concentrations than would be attained by uniform distribution in all brain cell types. We think that a local cellular distribution is more likely. In conclusion, following injection of [l25I]PEG-SOD, brain-associated levels of this radiolabeled enzyme are low. Following trauma, there is a 6- to 10-fold elevation of brain-associated PEG-SOD. The exact cellular location at which the PEG-SOD becomes concentrated in the brain is uncertain. Future experiments should be

that brain-associated PEG-SOD is very low in control animals. However, was

90

BRAIN SOD AFTER INJURY

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aimed at determining the cellular localization of therapeutic doses of PEG-SOD. It is only through determination of the site of action of PEG-SOD that insight into the local mechanisms that induce free radical damage will be understood.

ACKNOWLEDGMENTS This work was supported by NIH grants NS27214 and NS12587. E.F. Ellis is the Neuroscience Investigator Award.

recipient

of

a

Javits

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91

YOSHIDA ET AL.

YOSHIDA, K., BURTON, G.F., McKINNEY, J.S.,

glycol-conjugated Superoxide

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ZHANG, X.-M., and ELLIS, E.F. (1991). Superoxide dismutase decreases mortality, blood pressure and cerebral blood flow responses induced

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Address

reprint requests

to:

Dr. Earl F. Ellis Box 613, MCV Station Richmond, VA 23298-0613

92

Brain levels of polyethylene glycol-conjugated superoxide dismutase following fluid percussion brain injury in rats.

Polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) is being explored as an agent to reduce oxygen radical-mediated damage following brain i...
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