Influence of Polyethylene Glycol Superoxide Dismutase/Catalase on Altered Opioid-induced Pial Artery Dilation after Brain Injury

Newborn pigs (1–5 days old) of either sex were used in these experiments. All protocols have been approved by the Institutional Animal Care and Use Committee. They were sedated with 33 mg/kg intramuscular ketamine hydrochloride and 3.3 mg/kg intramuscular acepromazine. Anesthesia was maintained with alpha-chlorose (30–50 mg/kg, supplemented with 5 mg *symbol* kg sup -1 *symbol* h sup -1 intravenous). A catheter was inserted into a femoral artery to record blood pressure and to sample for blood gases and pH levels. A second catheter was placed into the femoral vein for drug administration. The trachea was cannulated and the animal's lungs were ventilated mechanically with room air. Body temperature was maintained at 37–38 degrees C with a heating pad.

For insertion into the cranial window, the scalp was removed and an opening was made in the skull over the parietal cortex. The dura mater was cut and retracted over the cut bone edge. The cranial window was placed in the hole and cemented in place with dental acrylic. The space under the window was filled with artificial CSF of the following composition (in mM): 3.0 KCl, 1.5 MgCl², 1.5 CaCl², 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 NaHCo³/liter, pH 7.33, P^CO2, 46 mmHg, and P^O243 mmHg. [10]The artificial CSF was warmed to 37–38 degrees C before application to the cerebral cortical surface.

Pial arteries were observed with a dissecting microscope, a television camera mounted on the microscope, and a video monitor. Vascular diameter was measured with a video microscaler (model VPA 550, For-A-Corp., Los Angeles, CA).

Methods for FPI have been published previously. [4]Briefly, a small hole was made in the skull contralateral to the cranial window, with the dura mater left intact. The opening was connected to a right angle metal shaft. The cranial window and the metal shaft were sealed in the skull with dental acrylic. The metal shaft was connected to a transducer housing and this in turn connected to the fluid percussion device. The device itself consisted of a methyl methacrylate polymer cylindrical reservoir 60-cm long, 4.5 cm in diameter, and 0.5-cm thick. One end of the device was connected to the transducer housing while the other end had a methyl methacrylate polymer piston mounted on O-rings. The exposed end of the piston was covered with a rubber pad. The entire system was filled with 0.9% saline (37 degrees C). The percussion device was supported by two brackets mounted on a metal platform. Brain injury was induced by striking the piston with a 4.8-kg pendulum. Intensity of the blow (usually 1.9–2.3 atm with a constant duration of 19–23 ms) was modified by varying the height from which the pendulum was allowed to fall. The pressure pulse was recorded on a storage oscilloscope triggered photoelectrically by a sensor activated by the fall of the pendulum. The amplitude of the pressure pulse was used to determine the intensity of the injury.

Pial artery diameter (small artery diameter 120–160 micro meter; arteriole diameter 50–70 micro meter) was determined every minute for a 10-min exposure period after injection under the window of artificial CSF containing no drug and after injection of CSF containing a drug. Typically, 1–2 ml CSF was flushed through the window over 30 s. Needles incorporated into the side of the window allowed infusion of CSF under the window and removal of CSF from under the window. We measured the peak response and a CSF sample for cGMP analysis was collected at the end of the 10-min exposure period. Cerebral cortical periarachnoid CSF (300 micro liter) was collected by slowly infusing CSF into one side of the window and allowing the CSF to drip freely into a collection tube on the opposite side.

Superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of superoxide anion generation using methods published previously. [14,17,18]SOD-inhibitable NBT reduction was determined during the first 20 min after a 60-min waiting period subsequent to FPI in piglets treated with vehicle (saline) and in piglets pretreated with PEGSOD (1,000 U/kg, Sterling Winthrop, Collegeville, PA) and catalase (10,000 U/kg, Sigma Chemical, St. Louis, MO) 30 min before FPI. In a third group of pigs, SOD-inhibitable NBT reduction was determined for 20 min without prior FPI. SOD-inhibitable NBT reduction was determined by placing NBT (Sigma, 2.4 mM) dissolved in CSF under one window and NBT (2.4 mM)+ SOD (Sigma, 60 U/ml) in CSF under the other 60 min after FPI. Both windows were located contralateral to the adapter for the induction of brain injury. NBT is water-soluble and forms a yellow solution that is converted to nitroblue formazan, an insoluble purple precipitate, in the presence of reducing agents. The SOD-inhibitable NBT reduction was determined by the difference in the quantities of nitroblue formazan precipitated on the brain surface under the two windows. Although NBT can be reduced by a variety of agents, SOD provides specificity for the assay.

To determine the quantity of precipitated nitroblue formazan, 1-mm thick slices of the brain surface under each cranial window were removed. For analysis, the slices were minced and homogenized in 1 N NaOH and 0.1% sodium dodecyl sulfate, the mixture centrifuged at 20,000g for 20 min, the resulting supernatant discarded, and the pellet resuspended in 3 ml pyridine. The formazan was dissolved in the pyridine during heating at 80 degrees C for 1 h. Particulate matter was removed by a second centrifugation at 10,000g for 10 min. The concentration of nitroblue formazan in the resultant solution was determined spectrophotometrically at 515 nm. The nitroblue formazan on the NBT alone side was read against the background of the SOD-treated side. Freshly prepared calibration solutions were used with each set of samples and treated identically to the samples.

Responses to methionine enkephalin, leucine enkephalin, dynorphin (10 sup -10, 10 sup -8, 10 sup -6 M, Sigma), DAMGO, DPDPE (10 sup -8, 10 sup -6 M, Research Biochemicals Int. Natick, MA), Deltorphin II (10 sup -8, 10 sup -6 M, Bachem, Torrance, CA) and U50, 488H (10 sup -8, 10 sup -6 M, Upjohn, Kalamazoo, MI) were obtained before and 1 h after FPI. A maximum of three drugs were investigated in each animal. Each drug was topically applied in an ascending concentration manner and 20 min was allowed after the highest concentration was washed off the cortical surface before a new drug was administered. Pretreatment with intravenous PEGSOD/catalase (1,000 U/kg and 10,000 U/kg, respectively) occurred after obtaining control responses to the opioids in question. A 30-min equilibration period was allowed after administration of SODCAT before FPI. All working drugs were made fresh on the day of use. The vehicle for all drugs (0.9% saline) had no effect on pial artery diameter (132 +/-4 vs. 133+/-4 micro meter, n = 5). Time control experiments were designed to obtain responses to agents initially and then again 60 min later.

Cerebrospinal fluid samples collected after a 10-min exposure to a drug were analyzed for cGMP using scintillation proximity assay methods. Commercially available kits for cGMP (Amersham, Arlington Heights, IL) were used. Briefly, this assay determines cyclic nucleotide concentration for binding to an antiserum that has a high specificity for cGMP. The antibody-bound cyclic nucleotide is then reacted with an antirabbit second antibody bound to fluoromicrospheres. Labeled cyclic nucleotide bound to the primary rabbit antibody can then be measured by determining the amount of light emitted by the fluoromicrospheres. All unknowns were assayed at two dilutions. The concentration of unlabeled cyclic nucleotides is calculated from the standard curve via linear regression analysis.*.

Pial arteriolar diameter, systemic arterial pressure, and cyclic nucleotide levels were analyzed using analysis of variance for repeated measures. If the value was significant, Fisher's exact test was performed. An alpha level of P < 0.05 was considered significant in all statistical tests. The n values reflect data for one vessel in each animal. Values are represented as mean+/-standard error of absolute values or as percentages of change from control values. Data presented as percent change were compared by nonparametric means using the Wilcoxon signed rank test.

Superoxidase dismutase-inhibitable NBT reduction was increased after brain injury and this increase was blunted by pretreatment with SODCAT (Figure 1).

Methionine enkephalin, a micro agonist (10 sup -10, 10 sup -8, 10 sup -6 M) elicited reproducible pial small artery (120–160 micro meter) and arteriole (50–70 micro meter) vasodilation (Table 1). This increase in vessel diameter was attenuated after brain injury and partially restored by SODCAT (Figure 2). Methionine enkephalin (10 sup -8, 10 sup -6 M) produced dilation that was associated with increased cortical periarachnoid CSF cGMP and these biochemical changes were blunted by brain injury and partially restored by SODCAT (Figure 3). These values represent a 1.5+/-0.1 and 1.9+/-0.1-fold change in CSF cGMP for methionine enkephalin 10 sup -8, 10 sup -6 M, respectively before injury versus 1.1+/-0.1 and 1.2+/-0.1-fold change after FPI versus 1.4+/-0.1 and 1.7+/-0.1-fold change in cGMP for FPI animals pretreated with SODCAT. Methionine enkephalin (10 sup - 10M), a physiologic concentration, also increased CSF cGMP and this response was partially restored by SODCAT (367+/-21 vs. 507 +/-225 and 296+/-3 vs. 417+/-23 fmol/ml).

Leucine enkephalin, a delta agonist, (10 sup -10, 10 sup -8, 10 sup -6 M), also produced pial artery dilation (Table 1) that was similarly attenuated after brain injury and partially restored by SODCAT (Figure 4). Leucine enkephalin-induced dilation also was associated with an increase in CSF cGMP, which was attenuated after brain injury and partially restored by SODCAT (Figure 3). These values represent a similar change in CSF cGMP as compared with methionine enkephalin.

In contrast, dynorphin, a kappa agonist, (10 sup -10, 10 sup -8, 10 sup -6 M) increased pial artery diameter (Table 1), which was reversed to vasoconstriction after brain injury and was restored to a vasodilator by SODCAT (Figure 5). Dynorphin (10 sup -8, 10 sup -6 M) produced dilation that was associated with a large increase in CSF cGMP. These biochemical changes were blunted after brain injury and partially restored by SODCAT (Figure 6). These values for dynorphin, 10 sup -8, 10 sup -6 M, represent a 1.9+/-0.1 and 2.9+/-0.1-fold change in CSF cGMP before injury versus 1.1+/-0.1 and 1.1 +/-0.1-fold change after brain injury versus 1.7+/-0.1 and 2.5+/-0.1-fold change after FPI for animals pretreated with SODCAT. Similar to methionine enkephalin, a physiologic concentration of dynorphin (10 sup -10 M) also increased CSF cGMP and this response was partially restored by SODCAT (373+/-18 vs. 541+/-13 and 298+/-3 vs. 438+/-15 fmol/ml).

Inasmuch as the above endogenous opioids may interact with several opioid receptor subtypes, synthetic analogs also were used to characterize the influence of brain injury on opioid-mediated dilation. DAMGO, a selective micro agonist, (10 sup -8, 10 sup -6 M) elicited reproducible small pial artery and arteriole vasodilation (Table 2), which was attenuated by FPI and partially restored by SODCAT (Figure 7). DAMGO-induced dilation was associated with an increase in CSF cGMP, which was attenuated after brain injury and partially restored by SODCAT (Figure 8). DPDPE and deltorphin, selective delta¹, and delta²opioid agonists, respectively (10 sup -8 M, 10 sup -6 M), also increased pial artery diameter (Table 2). These increases in diameter were attenuated by brain injury and partially restored by SODCAT (Figure 9). DPDPE- and deltorphin-induced dilation were associated with increases in CSF cGMP, which were attenuated after brain injury and partially restored by SODCAT (Figure 10).

Similar to dynorphin, U50,488H, a selective kappa agonist (10 sup -8, 10 sup -6 M), increased pial artery diameter (Table 2), which reversed to vasoconstriction after FPI and was restored to a vasodilator by SODCAT (Figure 7). Dilation induced by U50,488H was associated with an increase in periarachnoid cortical CSF cGMP, which was attenuated after brain injury and partially restored by SODCAT (Figure 8).

In five piglets, FPI decreased small pial artery diameter from 141 +/-4 to 122+/-5 and pial arterioles from 68+/-1 to 53+/-2 micro meter 1 h after injury. On a percentage basis, these changes reflect a 14+/-3 and 22+/-3 decrease in small artery and arteriole diameter, respectively, 1 h after FPI and diameter remained depressed thereafter. In contrast, brain injury only decreased small pial artery diameter from 147+/-5 to 132 +/-5 micro meter and arteriole diameter from 59+/-3 to 54 +/-1 micro meter in SODCAT-pretreated animals. On a percentage basis, these changes reflect a 9+/-1 and 10+/-1% decrease in small pial artery and arteriole diameter, respectively. Similarly, brain injury decreased resting control values for cGMP, and SODCAT pretreatment partially prevented those decreases (Figure 3, Figure 6, Figure 8, and Figure 10). Additionally, brain injury also decreased mean arterial blood pressure from 70+/-2 to 60 +/-2 mmHg and SODCAT pretreatment partially prevented this decrease (69+/-2 to 64+/-2 mmHg).

Blood chemistry values were obtained at the beginning and end of all experiments as well as immediately after brain injury. These values were: 7.40+/-0.01, 33+/-1, and 91+/-2 versus 7.40 +/-0.03, 35+/-3, and 88+/-4 versus 7.41 +/-0.05, 32+/-1, and 89+/-2 for pH, P^CO2, and P^O2before injury, after brain injury, and at the end of the experiment, respectively, n = 37. Mean level of brain injury was 2.1 +/-0.1 atm.

Results of the current study show that FPI increases SOD-inhibitable reduction of NBT by newborn pig brains, indicating that superoxide anion radical is generated. Pretreatment with SODCAT blunted the increase in SOD-inhibitable reduction of NBT associated with FPI. The endogenous opioids methionine enkephalin and leucine enkephalin, modestly selective micro and delta opioid agonists, respectively, [19]were associated with pial artery vasodilation and increased cortical periarachnoid CSF cGMP and these biochemical changes were attenuated by brain injury. In contrast, pretreatment with SODCAT partially restored the opioid-induced dilator responses and ability to increase CSF cGMP after brain injury. Moreover, dynorphin, an endogenous kappa agonist, was reversed from a dilator to a constrictor after FPI and this response was restored to a vasodilator by SODCAT. Because the lower concentration of endogenous opioids investigated in this study represent a physiologic CSF concentration present after brain injury, [10,12]data from the present study support previous research [13]that responses to methionine enkephalin and leucine enkephalin were attenuated while dynorphin was reversed from a dilator to a constrictor and thereby contributes to pial vasoconstriction after brain injury.

Although the endogenous opioids methionine enkephalin, leucine enkephalin, and dynorphin have recently been shown to be rather selective agonists for micro, delta, and kappa receptors, respectively, in the piglet cerebral circulation, [20,21]others consider them to be quite promiscuous in their receptor interactions. Therefore, synthetic analogs more selective for the opioid receptor subtypes were also used in this study to lend pharmacologic support to the physiologic findings of the endogenous opioids. Accordingly, it was observed that brain injury-induced attenuation of pial dilation and associated changes in CSF cGMP produced by the opioid receptor analogs DAMGO, DPDPE, and deltorphin, selective micro, delta¹, and delta²agonists, respectively, [22,23,24]was partially restored by pretreatment with SODCAT. Similar to dynorphin, brain injury also reversed the synthetic kappa agonist, U50,488 H, [25]from a vasodilator to a vasoconstrictor, this response being restored to a dilator by pretreatment with SODCAT.

It has been shown that superoxide anion is generated on the cerebral cortex of the newborn pig after bicuculline-induced seizures, [18]postischemic reperfusion, [17]asphyxia/reventilation, [26]and after placement of extravascular blood on the brain. [27]The amount of SOD-inhibitable NBT reduction generated in the current study after brain injury (14+/-2 pmol/mm²in 20 min) was much greater than that seen for bicuculline-induced seizures and ischemia/reperfusion (2.4 +/-0.6 pmol/mm²in 20 min and 8.7+/-1.5 pmol/mm²in 20 min for bicuculline-induced seizures and ischemia/reperfusion, respectively), although comparable to that seen for asphyxia/reventilation and extravascular blood (14.7+/-4.5 pmol/mm²in 20 min and 15.3+/-6 pmol/mm²in 20 min for asphyxia/reventilation and extravascular blood, respectively). The cerebrovascular consequences of the quantity of free radical production are not fully understood. However, there is a significant amount of evidence that supports a role of oxygen radicals in brain injury. For example, brain injury has been reported to cause the generation of superoxide for at least 1 h after injury. [28]In that study, the sustained dilation and abnormal responsiveness of pial arterioles observed after injury could be reversed by treatment with the free radical scavengers SOD and catalase. [28]Oxygen radicals also have been shown to increase blood-brain barrier permeability, [29,30]produce ultra-structural changes in pial vessel endothelium, [31]and cause abnormal arteriolar reactivity. [31]In addition, oxygen radical scavengers have been shown to improve vascular function and blood flow during focal ischemia in rats, which may account for the observed reductions in infarct size. [32]Recently, a trial with SOD in humans with severe head injuries showed that death and vegetative state was increased in patients receiving a placebo, as compared to those receiving PEGSOD. [16]In the heart, the antioxidant system is not fully mature at birth. [33]If we assume that the developing brain also has an immature antioxidant system, we can speculate that superoxide anion generated after FPI in the newborn pig has the potential to cause tissue damage. Accordingly, SODCAT pretreatment in the current study may supplement the endogenous immature antioxidant system, resulting in partial restoration of altered responses after brain injury.

The mechanism for the altered cerebral hemodynamics observed after brain injury has been investigated previously. For example, functional alterations have been accompanied by abnormalities in endothelial morphology and impairment of endothelium-dependent relaxation, [4,5]suggesting that altered release of endothelium-derived relaxing factor contributes to the reduction in cerebral blood flow after brain injury. Recently, it has been observed that nitric oxide, an example of an endothelium-derived relaxing factor, contributes to opioid-induced pial artery dilation. [34]In the newborn pig, opioids appear to play an important role in the control of the cerebral circulation. For instance, CSF concentrations of opioids are in the vasoactive range under control conditions and opioids contribute to the vascular actions of physiologic stimuli such as hypoxia and hemorrhagic hypotension. [8,20,21]Intracellular generation of superoxide anion or other species could alter nucleotides, second messengers, receptors, and membranes and the movement of superoxide anion out of the cell through anion channels could result in high concentrations of activated oxygen species at cell surfaces, including endothelium. Such oxygen species, then, may alter opioid-related nitric oxide generation, metabolism, or action. The blood-brain barrier normally limits entry of SOD into the brain parenchyma. Nevertheless, extracellular SOD is thought to protect against cellular damage from toxic oxygen species by consuming superoxide anion in the extracellular space, thereby promoting rapid concentration-dependent diffusion of superoxide anion out of the intracellular space. Interestingly, it was observed recently that SOD penetrates well into brain tissue after injury. [35]Recent experiments with a cell culture model of traumatic brain injury suggest that SOD reduces injury to stretched endothelial cells. [36]Results of the current study suggest that PEGSOD and catalase act on the cerebral vasculature of the newborn pig to improve altered vasculature reactivity after brain injury. Additionally, new data from this study also show that pial vessels do not constrict as greatly after brain injury in animals pretreated with SODCAT. The dose of SODCAT used in the current vascular reactivity studies was chosen based on the observation that this dose was sufficient to block superoxide production observed after brain injury (Figure 1). These data, therefore, further suggest that superoxide anion contributes to altered opioid-induced cerebrovascular effects after brain injury possibly via interference with nitric oxide.

While many techniques have been used experimentally to investigate brain injury, it has been suggested that the lateral fluid percussion technique used in the current study may model many of the sequelae associated with closed head injury in the human. [37]Previous studies have characterized the hemodynamic effects of brain injury in adult animals. [3,4,38]However, few studies have investigated the effects of brain injury in the newborn/infant time period. The present study may approximate the human newborn/infant time period because both newborn pigs and children younger than 1 yr have skulls with unfused sutures. It has been observed that developmental changes result in markedly different effects of brain injury on cerebral hemodynamics in the newborn and juvenile pig. [11]For example, it was observed that pial vessels constricted more and regional blood flow decreased and remained depressed longer in newborns vs juveniles. Furthermore, systemic arterial pressure has been observed to increase in the adult studies, [4]and in juvenile pigs, [11]whereas results of the current study show that systemic arterial pressure decreases after brain injury in the newborn pig, consistent with previous newborn studies. [11,12]Therefore, cerebral and systemic hemodynamic responses after brain injury are age-dependent.

The role of the systemic pressor response in altered adult cerebral hemodynamics has been investigated. For example, it was hypothesized that acute elevations of blood pressure after injury in the adult result in the release and metabolism of arachidonic acid, which would generate oxygen free radicals, causing cerebral functional abnormalities. [4,5,28,35,39]Results of the current study confirm that superoxide is generated 1 h after FPI in the newborn pig. However, in contrast to studies performed in adult and juvenile animals, there was no acute elevation in blood pressure after injury, which corroborates previous studies in the newborn pig. [11,12]In contrast, brain injury results in a decrease in mean arterial pressure, which remains depressed for up to 3 h after injury. [11,12]Of note, the distinction between previous data in the adult and the current study is that superoxide anion is only generated after an acute elevation in the adult (blockade of elevation in blood pressure blocks superoxide generation), whereas superoxide is released despite no such elevation, indeed, a depression in blood pressure in the newborn pig.

Opioids themselves also have been investigated for their contributory role in the cerebral hemodynamic effects of brain injury. For example, the opioid antagonist naloxone has been observed to improve blood chemistry, electroencephalographic parameters, and brain perfusion pressure in cats, [40]attenuate pial artery constriction and reductions in cerebral blood flow after brain injury in piglets, [12]and improve long-term neurobehavioral outcome after brain injury in rats. [41]Regional increases in dynorphin immunoreactivity in the parietal and frontal cortex, pons, medulla, and striatum were found to correlate with local histopathologic damage and reductions in cerebral blood flow after brain injury in the adult cat, [42]suggesting that dynorphin and the kappa opioid system could play a role in the injury process after brain trauma. Further evidence in support of this concept is found in the observations that administration of dynorphin or the synthetic kappa agonist, U50,488 H, worsens neurologic outcome, [43]whereas the kappa antagonist, nalmafene, improves neurologic outcome and metabolism after brain injury. [44]Alternatively, U50,488 H also has been reported to improve spinal cord blood flow and neurologic recovery after brain injury in mice. [45]A partial explanation for these contradictory data could be that there are different kappa isoreceptors that mediate different physiologic effects and the above drugs could have varying affinities for these receptors. [43]Moreover, methionine enkephalin (a micro agonist) release could be neuroprotective as micro and kappa opioid receptor concentrations could be regulated differently after brain injury. [43,46]Recent studies in the newborn pig show that CSF opioid concentrations increase after brain injury and that the time course and relative increase in CSF concentration vary from opioid to opioid. [12]Data from a recent study [13]suggest that methionine enkephalin and leucine enkephalin, endogenous micro and delta agonists, respectively, produce pial dilation that would be beneficial, serving as a physiologic antagonist to brain injury and pial artery constriction. Although the CSF concentration of these two opioids is increased after brain injury, their beneficial role is decreased because dilation by these opioids is attenuated after brain injury. [13]Moreover, brain injury reversed dynorphin from a dilator to a constrictor, further contributing to pial artery vasoconstriction after injury. Although the mechanism for this reversal is uncertain, there are several possibilities. First, dynorphin is a tone-dependent agent eliciting dilation during normotension and constriction during hypotension. [6]Because brain injury produces modest hypotension in the newborn pig, this could contribute to the reversal of dynorphin from a dilator to a constrictor. In addition, brain injury-induced alteration of nitric oxide function, as demonstrated in the current study, could unmask a different kappa isoreceptor or smooth muscle kappa constrictor component of dynorphin. Finally, although opioids were the only vasoactive substances investigated in the current study, it should be emphasized that brain injury-related altered vascular responsiveness is probably not selective for opioids. For example, it has recently been observed that brain injury reverses vasopressin from a dilator to a constrictor after injury. [47].

In conclusion, results of the current study show that superoxide anion is produced after brain injury. In addition, opioid-induced pial artery vasodilation and associated changes in CSF cGMP are attenuated after brain injury and these changes are partially restored after brain injury by PEGSOD and catalase. Further, these data suggest that superoxide anion contributes to altered opioid induced cerebrovascular effects after brain injury, suggesting a possible beneficial role for free radical scavenger use with traumatic brain injury.