JOURNAL OF NEUROTRAUMA Volume 9, Number 4, 1992 Mary Ann Liebert, Inc., Publishers
Assessment of Posttraumatic Polymorphonuclear Leukocyte Accumulation in Rat Brain Using Tissue Myeloperoxidase Assay and Vinblastine Treatment KATHERINE V.
BIAGAS,1 MARK W. UHL,' JOANNE K. SCHIDING,1 NEMOTO,1 and PATRICK M. KOCHANEK1'2
EDWIN M.
ABSTRACT
Polymorphonuclear leukocytes (PMN) are implicated in the pathogenesis of traumatic brain injury. We tested the following hypotheses: (1) leukocyte accumulation is present in brain tissue 24 h posttrauma, (2) leukocyte accumulation represents PMN, and (3) prior systemic PMN depletion attenuates brain tissue PMN accumulation. Trauma was induced in exposed right parietal cortex by weightdrop in anesthetized Wistar rats (n 24). Of the traumatized rats, 12 were PMN-depleted with vinblastine sulfate i.v. Controls were 12 normal rats and 5 sham-operated rats (craniotomy). Sections of traumatized and contralateral hemispheres were analyzed for myeloperoxidase (MPO) activity. Brain MPO activity was increased fivefold at 24 h posttrauma, but only in the traumatized hemisphere (0.448 ± 0.133 U/g vs 0.090 ± 0.022 U/g in trauma vs normal, respectively, p < 0.05, mean ± SEM). PMN depletion attenuated this increase in MPO activity and decreased circulating PMN counts (0.07 ± 0.032 x 109/L vs 0.894 ± 0.294 x 109/L PMN-depleted-trauma vs trauma rats, respectively, p < 0.05). Leukocyte accumulation in the brain posttrauma was confirmed by MPO assay. Inhibition of MPO activity in the PMN-depleted group and the specificity of vinblastine treatment for depletion of circulating PMN suggest that leukocyte accumulation in the brain at 24 h posttrauma is largely due to PMN. =
INTRODUCTION leukocytes (PMN) have been implicated in the pathogenesis of traumatic brain for PMN are postulated in blood-brain barrier injury, edema formation, and roles injury. Important disturbances in cerebral blood flow (Hallenbeck et al., 1986; Schoettle et al., 1990; Faraci et al., 1991 ). These roles, however, are speculative in traumatic injury. We previously reported accumulation of "'in-labeled PMN during the first 8 h after percussive trauma in rats (Schoettle et al., 1990). Accumulation 2 h after trauma
Polymorphonuclear
'Department of Anesthesiology and Critical Care Medicine and 2 Department of Pediatrics, University of Pittsburgh, Pittsburgh. Pennsylvania. 363
BIAGAS ET AL.
explained by an increase in cerebral blood volume. However, accumulation between 4 and 8 h posttrauma occurred despite a reduction in cerebral blood volume and represented acute inflammation. PMN accumulation at both times correlated with the amount of posttraumatic cerebral edema observed. Difficulties exist, however, with the use of either conventional histology or labeled PMN to quantitate PMN accumulation in brain, particularly in determining early postinsult accumulation. Del Zoppo et al. (1991) assessed intravascular PMN accumulation early after carotid artery occlusion. However, the information obtained was semiquantitative, and the technique used required an elaborate protocol with extensive tissue sectioning. Similarly, methods using labeled PMN require isolation and manipulation of PMN, which may trigger activation mechanisms and alter in vivo activity (Rinaldo et al., 1988). Ideally, the PMN response to injury should be examined without direct manipulation of the leukocytes. Myeloperoxidase (MPO), a lysosomal enzyme specific to leukocyte granules, has been used as a marker of PMN accumulation in models of inflammation or injury in skin, intestine, liver, lung, and spinal cord (Bradley et al., 1982; Krawiszetal., 1984; Xu et al., 1990). However, in vitro studies have shown that MPO activity can be blocked by several inhibitors (Clark and Klebanoff, 1979). Tissue inhibitors of MPO have been identified in liver and kidney and are postulated to interfere with brain MPO activity as well (Duval et al., 1990;HillegassetaL, 1990; Barone et al., 1991). Barone et al. (1991) modified the standard MPO assay for use in brain tissue. In their modification, brain tissue homogenates were washed before MPO extraction. Using this assay, increased MPO activity was demonstrated in rat brain 24 h after middle cerebral artery occlusion and reperfusion. This increase was attributed to PMN accumulation. Often portrayed as a specific PMN marker, MPO is found in PMN, monocytes, and macrophages, although, monocytes contain only as much as one third of the MPO activity contained in PMN (Bos et al., 1978). In this study, we used the MPO assay in a model of focal cerebral trauma in rats and examined endogenous brain tissue MPO activity 24 h posttrauma as a means of assessing PMN accumulation. We examined the MPO response 24 h after the injury because peak edema was observed at 24 h posttrauma (Grundl et al., 1991), and clinically relevant disturbances in brain water and blood flow are observed at 24-48 h after traumatic injury (Obrist et al., 1979; Bruce et al., 1979). The hypotheses tested were ( 1 ) increased MPO activity is present in brain tissue 24 h posttrauma, (2) MPO activity represents PMN rather than monocyte accumulation, and (3) systemic PMN depletion with vinblastine sulfate before trauma attenuates PMN accumulation. was
MATERIALS AND METHODS Cerebral Trauma Model and Tissue
Preparation
approved by the University of Pittsburgh Animal Care and Use Committee. Forty-one Wistar rats (older than 2 months, weighing 350-450 g) had free access to food and male virus-free, mature of the until the time water study. The rats were assigned to one of four groups: ( 1 ) trauma without vinblastine pretreatment (n 12), (2) trauma with vinblastine pretreatment 5 days before trauma (n 12), (3) normal without trauma or pretreatment in which portions of brain from each rat were PMN enriched (n 12), and (4) sham (craniotomy only) without vinblastine pretreatment (n 5). Anesthesia was induced in a plastic jar with 4% halothane in oxygen. The trachea was intubated with a 14-gauge angiocatheter, and the lungs were mechanically ventilated with 1 % halothane/66% N20/33% 02. A femoral venous catheter was inserted, using aseptic technique, for the administration of pancuronium bromide (0.1 mg kg" 'h~ ', Elkins-Sinn, Cherry Hill, NJ). A femoral arterial catheter was similarly inserted for continuous monitoring of blood pressure. A rectal probe was inserted for continuous monitoring and maintenance of temperature at 37.5°C ± 0.5°C, with the aid of a heated water blanket. Bicillin (100,000 U, Upjohn, Kalamazoo, MI) and gentamicin (10 mg/kg, Elkins-Sinn, Cherry Hill, NJ) were administered intramuscularly. Fifteen minutes before trauma, an arterial blood sample (0.4 ml) was obtained to verify that blood gas values and hematocrit were within normal limits. Traumatic brain injury was performed in 24 rats as previously described (Schoettle et al., 1990). After craniotomy, percussive injury to the right parietal cortex was induced by dropping a 10 g brass rod (3 mm in All studies
were
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BRAIN MYELOPEROXIDASE ACTIVITY AFTER TRAUMA
diameter) from a height of 5 cm through a glass guide tube onto the exposed surface of the brain. Sham insult
(craniotomy with removal and replacement of the bone flap) was performed in 5 rats. Within
1 h of surgery, all rats were extubated and returned to their cages. The rats were reanesthetized as previously described 24 h after trauma or sham. Through a midline thoracotomy, the aorta was clamped at the midthorax level. To eliminate intravascular blood components, the head and upper torso were perfused transcardially with 350 ml of isotonic saline infused into the left ventricle. Central venous perfusate became clear after perfusion of approximately 200 ml. Normal rats were anesthetized with halothane and transcardially perfused as described. Brains were removed quickly, and 4 mm thick sections (150-300 mg in weight) of the trauma region and corresponding contralateral hemisphere were excised and weighed. Each brain tissue section was homogenized in 4 ml of 50 mM potassium phosphate buffer, pH 6.0 (Sigma, St. Louis, MO), and was centrifuged at 2500g for 10 min. The supernatant was discarded, and the pellet was resuspended in 10 ml of potassium phosphate buffer. Centrifugation and suspension in buffer were repeated twice to dilute inhibitors of MPO activity (Barone et al., 1991). Homogenates were pelleted and assayed for MPO activity.
Preparation of PMN-Depleted Rats Five days before trauma, 12 rats were anesthetized, femoral venous catheters were inserted, and 0.5 mg/kg of vinblastine sulfate was administered i.v. Bicillin (100,000 U) and gentamicin (10 mg/kg) were given i.m. to prevent infection. The catheters were removed, and the rats were allowed to recover and were returned to their cages. Trauma was performed 5 days later as described. For measurement of hématologie variables, complete blood counts (CBC) with leukocyte differential were performed in 18 rats randomly assigned to one of two groups: (1) CBC were obtained in 6 rats immediately before and 24 h after trauma to assess the effect of trauma on circulating PMN and monocytes, and (2) CBC were obtained in 12 vinblastine-treated rats 5 days (immediately before trauma) and 6 days (24 h after trauma) after vinblastine administration to assess the effect of vinblastine treatment on the circulating PMN count. The rats from which hématologie samples were obtained were separate from those used for MPO assessment in all but 3 cases in an attempt to avoid possible effects of acute blood loss from blood sampling on leukocyte accumulation.
Preparation of Isolated PMN and PMN-Enriched Brain
Tissue
as an internal control for the MPO assay, known amounts of isolated homologous rat PMN were for assayed MPO activity on each day that the MPO assay was performed. Conversion of the MPO signal in brain tissue into number of PMN was accomplished by adding known amounts of homologous PMN to brain samples from normal rats. Rat PMN were obtained from peritoneal exudates according to the method of Lemanske et al. (1983). Twenty-four rats not exposed to trauma and not used for brain tissue MPO determination were injected i.p. with casein (200 mg. Fisher, Fairlawn, NJ) in saline (5 ml). PMN-rich exudates were obtained 5 h later by intraperitoneal lavage with 20 ml of saline. Each exúdate was passed through clean gauze to remove particulate matter, and the leukocytes were counted with a hemocytometer. Purity was assessed using modified Wright's staining (Leuko-Stat, Fisher, Orangeburg, NY). Approximately 3 x 107 cells were obtained from each exúdate, and 91% ± 1% of the cells isolated were PMN. Serial dilutions of isolated PMN (1 x 104 to 1 x 106 cells) were then assayed for MPO activity. PMN-enriched brain homogenates were made by adding 2 X 104,5 x 104,andl X 105 isolated PMN to 1 ml aliquots of left hemisphere homogenates from control rats. Standard curves of MPO activity were generated for both PMN isolates and PMN-enriched brain homogenates.
To serve
MPO
Activity Assay
MPO activity was measured in brain tissue homogenates from normal rats and in rats 24 h after trauma or sham insult. MPO activity was also determined in isolated PMN and PMN-enriched brain from normal rats. 365
BIAGAS ET AL. Isolated PMN and brain tissue pellets were resuspended in 2.5 ml of hexadecyltrimethylammonium bromide (HTAB, 0.5% HTAB in 50 mM potassium phosphate buffer, pH 6.0, Sigma, St. Louis, MO). HTAB is a detergent that releases the MPO enzyme from leukocyte granules. Three freeze-thaw cycles were performed to further disrupt granules. Samples were centrifuged for 10 min at 2500g for a final time. Supernatant (100 p,l) was added to 2.9 ml of 50 mM potassium phosphate buffer with o-dianisidine hydrochloride (0.167 mg/ml, Sigma, St. Louis, MO) and H202 (0.0005%, Sigma, St. Louis, MO). Change in absorbance at 460 nm was assayed spectrophotometrically (Beckman, Somerset, NJ) for 2 min in triplicate. MPO activity was calculated as the mean of the three readings. One unit of MPO activity was defined as the degradation of 1 p.mole of H202/min (Barmen, 1969). Brain tissue results were expressed as units of MPO activity per gram of tissue. An estimate of accumulated PMN per gram of tissue was made from extrapolated MPO activity in PMN-enriched brains.
Statistical Analysis All data are presented as mean ± SEM. Brain tissue MPO activity in normal rats represents mean MPO activity of right and left hemispheric sections. Between-group comparisons of MPO activity in normal (n 12), sham (n 5), trauma (n 12), and PMN-depleted-trauma (n 12) rats were made 24 h posttrauma using one-way ANOVA and the Student-Newman-Keuls test. MPO activity in isolated PMN and in PMN-enriched brain tissue was analyzed by linear regression. Hématologie variables measured before and 24 h after trauma were analyzed in both trauma (n 6) and PMN-depleted-trauma (n 12) groups using Student's of /-test. paired Between-group comparisons hématologie variables were made using one-way ANOVA and Student-Newman-Keuls test. A p value less than 0.05 was considered to be statistically =
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significant. RESULTS All rats in all groups survived. As previously described in this model, rats recover, are extubated within 1 h, and are returned to their cages shortly thereafter (Schoettle et al., 1990). Physiologic variables, including arterial blood gases, mean arterial blood pressure, and rectal temperature, were measured in all rats at the time of surgery. There was no difference in any physiologic variable. Rats that were PMN-depleted before trauma differed from trauma rats in body weight, but all rats were within the predefined weight range of 350-450 g (430 ± 11 g vs 379 ± 6 g, PMN-depleted-trauma rats vs trauma rats, respectively, p < 0.05). Hématologie variables were measured in serial blood samples in a total of 18 rats ( 12 with PMN-depletion) before and 24 h after trauma (Table 1). An increase in total white blood cell count was observed 24 h posttrauma. This was due to increases in both absolute PMN and monocyte counts. No such increase in total white blood cell count was seen in vinblastine-treated rats. Vinblastine treatment markedly reduced the circulating absolute PMN count before trauma and 24 h posttrauma but did not decrease circulating monocyte or lymphocyte counts. Posttraumatic monocytosis occurred in nondepleted rats. In vinblastine-treated rats, the absolute monocyte count tripled, although this increase was not statistically significant. Hematocrit was slightly reduced by vinblastine treatment before and 24 h after trauma (p < 0.05) (Table 1). The reduction in hematocrit was 7% and should be expected to have minimal, if any, physiologic significance. A single 0.5 mg/kg dose of vinblastine had no effect on the circulating platelet count, as previously reported in our model (Uhl et al., 1992). MPO activity measured in isolated homologous PMN was linearly correlated to the number of PMN present (r2 0.99, p < 0.001)(Fig. 1). Brain tissue contains inhibitors of MPO activity that are mostly but perhaps not completely eliminated in the modified MPO assay (Barone et al., 1991). To equate the MPO signal to number of PMN in the brain, MPO activity was determined in PMN-enriched brains (Fig. 2). MPO activity was detected in all PMN-enriched brain aliquots. PMN count and MPO activity in PMN-enriched brain were linearly correlated (r2 0.96, p < 0.01) (Fig. 2). Tissue MPO activity in normal brain was 0.090 ± 0.022 U/g and between 0.060 U/g and 0.125 U/g in sham. In our study, MPO activity in normal rats was higher than previously reported in a model of spinal cord trauma using saline-perfused but nonwashed tissue (Xu et al., 1990). However, our results do concur with those of Barone et al. (1991), using a nearly identical technique in rat brain. =
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366
BRAIN MYELOPEROXIDASE ACTIVITY AFTER TRAUMA Table 1.
Hematologic Variables Measured 24 H After Trauma"
HCTb
T
(%)
V
WBC
t
(109/L)
V
ANC
T
(109/L)
V
AMC
T V T V
(109/L) ALC
(109/L)
Before
trauma
43.7 40.8 7.27 6.54 0.89 0.07 0.05 0.10 6.31 6.37
0.8 0.6** 0.49 0.58 0.29 0.03* 0.03 0.02 0.66 0.56
in
Rats Before
After 44.5 39.5 9.53 6.68 2.64 0.27 0.33 0.31 6.51 6.05
and
trauma
± ± ±
± ± ±
± ± ± ±
1.3 0.6** 0.43* 0.61 0.42* 0.10** 0.08* 0.10 0.64 0.57
"Rats received intravenous vinblastine sulfate or saline vehicle 5 days before Circulating hematocrit and leukocyte counts before and 24 h after trauma are shown. bT, trauma (n 6); V, vinblastine administered PMN-depleted-trauma (n 12); Hct, hematocrit; WBC, complete white blood count; ANC, absolute polymorphonuclear leukocyte count; AMC, absolute monocyte count; ALC, absolute lymphocyte count. *p < 0.05 before trauma vs after trauma. **p < 0.05 vinblastine administered vs trauma. trauma.
=
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MPO activity in the right hemisphere of the trauma group was different from MPO activity in either hemisphere of all other groups tested (p < 0.05) (Fig. 3). A fivefold increase in MPO activity was observed in sections from the injured hemisphere 24 h after trauma (0.448 ±0.13 U/g vs 0.090 ± 0.022 U/g in trauma vs normal, respectively, p < 0.0'j) (Fig. 3). No increase in MPO activity was observed in sections from the contralateral, uninjured hemisphere in trauma rats or sections from either hemisphere in sham rats. Rats depleted of PMN before trauma showed no increase in brain MPO activity in either the injured or contralateral hemisphere 24 h after trauma.
DISCUSSION
Supporting our primary hypothesis, we demonstrated a fivefold increase in brain tissue MPO activity 24 h after percussive trauma in rats. This information corroborates and augments previous work in this model using
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1
0.40 -0.3O
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-
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0.00
Control
R
L
R
L
R
L
Myeloperoxidase (MPO) activity (U/g) in normal (mean ± SEM of both hemispheres, Dn= 12), sham (craniotomy only,S«-= 5), injured (right) and uninjured (left) hemispheres in rats with trauma ( 0« 12), and rats with polymorphonuclear leukocyte (PMN)-depletion and trauma ( n 12). A fivefold increase in MPO activity is demonstrated in injured hemisphere of trauma rats. No increase in MPO activity is seen in either uninjured hemisphere or in injured hemisphere of rats rendered PMN-depleted before trauma. PMND, PMN-depleted. *p < 0.05 vs all other FIG. 3.
=
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groups.
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BRAIN MYELOPEROXIDASE ACTIVITY AFTER TRAUMA
increase in brain tissue MPO activity, suggests that the increase in brain tissue MPO 24 h posttrauma specifically represents PMN infiltration. It also suggests that a 93% reduction in the circulating absolute PMN count at the time of trauma prevents accumulation of PMN in brain tissue 24 h posttrauma. Using these observations and data extrapolated from the relation between PMN count and MPO activity in PMN-enriched brain tissue, we estimated PMN accumulation in traumatized tissue. A fivefold increase in MPO activity represents an accumulation of approximately 5 X 106 PMN/g brain tissue (about 1.5 X 106 PMN in each 300 mg section of injured brain tissue sampled in each rat) in the traumatized hemisphere during the first 24 h posttrauma. This study did not rule out a contribution of tissue macrophages (microglia) to the observed increase in brain MPO activity. As with monocytes, however, significant increases in the number of reactive microglia are not evident until 24 h or more posttrauma (Giulian et al., 1989). Furthermore, MPO activity in saline-perfused brain tissue from control rats, which would be expected to reflect tissue macrophages, was low but detectable. That MPO activity in the left hemisphere of vinblastine-treated rats was similar to control values suggests that a small level of non-PMN-generated MPO activity is present in brain tissue. Additional studies in rats not subjected to trauma would be needed to further address the source of this MPO activity. The mechanism(s) and location(s) of PMN accumulation were not addressed specifically in this study. However, saline perfusion of tissue should have eliminated free PMN in the vascular space, excluding them from MPO measurement. The remaining PMN are likely to be from several sources: (1) PMN infiltrating tissue, (2) PMN adhering to vascular endothelium, and (3) PMN in microthrombi and hemorrhage. These are all PMN that have pathologic location and play a potential role in local parenchymal and vascular injury. Such conclusions are supported by our previous work, in which PMN accumulation 4-8 h posttrauma exceeded changes in brain blood volume (Schoettle et al., 1990). If PMN or other leukocyte cell lines play important pathophysiologic roles in posttraumatic brain injury, recent advances in the ability to modify PMN accumulation and function by means of specific antibodies to adhesion receptors on both PMN and vascular endothelium may lead to significant improvements in therapy (Clark et al., 1991). In conclusion, a fivefold increase in brain PMN during the first 24 h after percussive trauma was demonstrated through the combined use of a recently modified MPO assay and selective PMN depletion produced by vinblastine sulfate in rats. This MPO assay can be used to assess the effects of various therapeutic interventions on posttraumatic PMN accumulation in the brain. In addition, depletion of circulating PMN attenuated posttraumatic PMN accumulation in the brain. Moreover, as effective PMN-endothelial-adhesion receptor antagonists are not readily available for the rat, PMN depletion with vinblastine is a potentially useful tool for the initial evaluation of the role of PMN in posttraumatic brain pathophysiology.
ACKNOWLEDGMENTS We acknowledge the generous support of a grant from the Brain Trauma Foundation and the American Heart Association, Pennsylvania Affiliate. We thank Drs. Frank Barone and Len Hillegass from SmithKline Beechman Research Laboratories and Dr. Heidi Horner from Athena Neurosciences for helpful input on the MPO assay. We thank Dr. Shekhar Venkataraman for critical review, Lisa Cohn for editorial assistance, and Janet Santucci, Carleen Heinz, and Becky Gmuer for preparation of this manuscript.
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371
Assessment of Posttraumatic Polymorphonuclear Leukocyte Accumulation in Rat Brain Using Tissue Myeloperoxidase Assay and Vinblastine Treatment KATHERINE V.
BIAGAS,1 MARK W. UHL,' JOANNE K. SCHIDING,1 NEMOTO,1 and PATRICK M. KOCHANEK1'2
EDWIN M.
ABSTRACT
Polymorphonuclear leukocytes (PMN) are implicated in the pathogenesis of traumatic brain injury. We tested the following hypotheses: (1) leukocyte accumulation is present in brain tissue 24 h posttrauma, (2) leukocyte accumulation represents PMN, and (3) prior systemic PMN depletion attenuates brain tissue PMN accumulation. Trauma was induced in exposed right parietal cortex by weightdrop in anesthetized Wistar rats (n 24). Of the traumatized rats, 12 were PMN-depleted with vinblastine sulfate i.v. Controls were 12 normal rats and 5 sham-operated rats (craniotomy). Sections of traumatized and contralateral hemispheres were analyzed for myeloperoxidase (MPO) activity. Brain MPO activity was increased fivefold at 24 h posttrauma, but only in the traumatized hemisphere (0.448 ± 0.133 U/g vs 0.090 ± 0.022 U/g in trauma vs normal, respectively, p < 0.05, mean ± SEM). PMN depletion attenuated this increase in MPO activity and decreased circulating PMN counts (0.07 ± 0.032 x 109/L vs 0.894 ± 0.294 x 109/L PMN-depleted-trauma vs trauma rats, respectively, p < 0.05). Leukocyte accumulation in the brain posttrauma was confirmed by MPO assay. Inhibition of MPO activity in the PMN-depleted group and the specificity of vinblastine treatment for depletion of circulating PMN suggest that leukocyte accumulation in the brain at 24 h posttrauma is largely due to PMN. =
INTRODUCTION leukocytes (PMN) have been implicated in the pathogenesis of traumatic brain for PMN are postulated in blood-brain barrier injury, edema formation, and roles injury. Important disturbances in cerebral blood flow (Hallenbeck et al., 1986; Schoettle et al., 1990; Faraci et al., 1991 ). These roles, however, are speculative in traumatic injury. We previously reported accumulation of "'in-labeled PMN during the first 8 h after percussive trauma in rats (Schoettle et al., 1990). Accumulation 2 h after trauma
Polymorphonuclear
'Department of Anesthesiology and Critical Care Medicine and 2 Department of Pediatrics, University of Pittsburgh, Pittsburgh. Pennsylvania. 363
BIAGAS ET AL.
explained by an increase in cerebral blood volume. However, accumulation between 4 and 8 h posttrauma occurred despite a reduction in cerebral blood volume and represented acute inflammation. PMN accumulation at both times correlated with the amount of posttraumatic cerebral edema observed. Difficulties exist, however, with the use of either conventional histology or labeled PMN to quantitate PMN accumulation in brain, particularly in determining early postinsult accumulation. Del Zoppo et al. (1991) assessed intravascular PMN accumulation early after carotid artery occlusion. However, the information obtained was semiquantitative, and the technique used required an elaborate protocol with extensive tissue sectioning. Similarly, methods using labeled PMN require isolation and manipulation of PMN, which may trigger activation mechanisms and alter in vivo activity (Rinaldo et al., 1988). Ideally, the PMN response to injury should be examined without direct manipulation of the leukocytes. Myeloperoxidase (MPO), a lysosomal enzyme specific to leukocyte granules, has been used as a marker of PMN accumulation in models of inflammation or injury in skin, intestine, liver, lung, and spinal cord (Bradley et al., 1982; Krawiszetal., 1984; Xu et al., 1990). However, in vitro studies have shown that MPO activity can be blocked by several inhibitors (Clark and Klebanoff, 1979). Tissue inhibitors of MPO have been identified in liver and kidney and are postulated to interfere with brain MPO activity as well (Duval et al., 1990;HillegassetaL, 1990; Barone et al., 1991). Barone et al. (1991) modified the standard MPO assay for use in brain tissue. In their modification, brain tissue homogenates were washed before MPO extraction. Using this assay, increased MPO activity was demonstrated in rat brain 24 h after middle cerebral artery occlusion and reperfusion. This increase was attributed to PMN accumulation. Often portrayed as a specific PMN marker, MPO is found in PMN, monocytes, and macrophages, although, monocytes contain only as much as one third of the MPO activity contained in PMN (Bos et al., 1978). In this study, we used the MPO assay in a model of focal cerebral trauma in rats and examined endogenous brain tissue MPO activity 24 h posttrauma as a means of assessing PMN accumulation. We examined the MPO response 24 h after the injury because peak edema was observed at 24 h posttrauma (Grundl et al., 1991), and clinically relevant disturbances in brain water and blood flow are observed at 24-48 h after traumatic injury (Obrist et al., 1979; Bruce et al., 1979). The hypotheses tested were ( 1 ) increased MPO activity is present in brain tissue 24 h posttrauma, (2) MPO activity represents PMN rather than monocyte accumulation, and (3) systemic PMN depletion with vinblastine sulfate before trauma attenuates PMN accumulation. was
MATERIALS AND METHODS Cerebral Trauma Model and Tissue
Preparation
approved by the University of Pittsburgh Animal Care and Use Committee. Forty-one Wistar rats (older than 2 months, weighing 350-450 g) had free access to food and male virus-free, mature of the until the time water study. The rats were assigned to one of four groups: ( 1 ) trauma without vinblastine pretreatment (n 12), (2) trauma with vinblastine pretreatment 5 days before trauma (n 12), (3) normal without trauma or pretreatment in which portions of brain from each rat were PMN enriched (n 12), and (4) sham (craniotomy only) without vinblastine pretreatment (n 5). Anesthesia was induced in a plastic jar with 4% halothane in oxygen. The trachea was intubated with a 14-gauge angiocatheter, and the lungs were mechanically ventilated with 1 % halothane/66% N20/33% 02. A femoral venous catheter was inserted, using aseptic technique, for the administration of pancuronium bromide (0.1 mg kg" 'h~ ', Elkins-Sinn, Cherry Hill, NJ). A femoral arterial catheter was similarly inserted for continuous monitoring of blood pressure. A rectal probe was inserted for continuous monitoring and maintenance of temperature at 37.5°C ± 0.5°C, with the aid of a heated water blanket. Bicillin (100,000 U, Upjohn, Kalamazoo, MI) and gentamicin (10 mg/kg, Elkins-Sinn, Cherry Hill, NJ) were administered intramuscularly. Fifteen minutes before trauma, an arterial blood sample (0.4 ml) was obtained to verify that blood gas values and hematocrit were within normal limits. Traumatic brain injury was performed in 24 rats as previously described (Schoettle et al., 1990). After craniotomy, percussive injury to the right parietal cortex was induced by dropping a 10 g brass rod (3 mm in All studies
were
=
=
=
=
364
BRAIN MYELOPEROXIDASE ACTIVITY AFTER TRAUMA
diameter) from a height of 5 cm through a glass guide tube onto the exposed surface of the brain. Sham insult
(craniotomy with removal and replacement of the bone flap) was performed in 5 rats. Within
1 h of surgery, all rats were extubated and returned to their cages. The rats were reanesthetized as previously described 24 h after trauma or sham. Through a midline thoracotomy, the aorta was clamped at the midthorax level. To eliminate intravascular blood components, the head and upper torso were perfused transcardially with 350 ml of isotonic saline infused into the left ventricle. Central venous perfusate became clear after perfusion of approximately 200 ml. Normal rats were anesthetized with halothane and transcardially perfused as described. Brains were removed quickly, and 4 mm thick sections (150-300 mg in weight) of the trauma region and corresponding contralateral hemisphere were excised and weighed. Each brain tissue section was homogenized in 4 ml of 50 mM potassium phosphate buffer, pH 6.0 (Sigma, St. Louis, MO), and was centrifuged at 2500g for 10 min. The supernatant was discarded, and the pellet was resuspended in 10 ml of potassium phosphate buffer. Centrifugation and suspension in buffer were repeated twice to dilute inhibitors of MPO activity (Barone et al., 1991). Homogenates were pelleted and assayed for MPO activity.
Preparation of PMN-Depleted Rats Five days before trauma, 12 rats were anesthetized, femoral venous catheters were inserted, and 0.5 mg/kg of vinblastine sulfate was administered i.v. Bicillin (100,000 U) and gentamicin (10 mg/kg) were given i.m. to prevent infection. The catheters were removed, and the rats were allowed to recover and were returned to their cages. Trauma was performed 5 days later as described. For measurement of hématologie variables, complete blood counts (CBC) with leukocyte differential were performed in 18 rats randomly assigned to one of two groups: (1) CBC were obtained in 6 rats immediately before and 24 h after trauma to assess the effect of trauma on circulating PMN and monocytes, and (2) CBC were obtained in 12 vinblastine-treated rats 5 days (immediately before trauma) and 6 days (24 h after trauma) after vinblastine administration to assess the effect of vinblastine treatment on the circulating PMN count. The rats from which hématologie samples were obtained were separate from those used for MPO assessment in all but 3 cases in an attempt to avoid possible effects of acute blood loss from blood sampling on leukocyte accumulation.
Preparation of Isolated PMN and PMN-Enriched Brain
Tissue
as an internal control for the MPO assay, known amounts of isolated homologous rat PMN were for assayed MPO activity on each day that the MPO assay was performed. Conversion of the MPO signal in brain tissue into number of PMN was accomplished by adding known amounts of homologous PMN to brain samples from normal rats. Rat PMN were obtained from peritoneal exudates according to the method of Lemanske et al. (1983). Twenty-four rats not exposed to trauma and not used for brain tissue MPO determination were injected i.p. with casein (200 mg. Fisher, Fairlawn, NJ) in saline (5 ml). PMN-rich exudates were obtained 5 h later by intraperitoneal lavage with 20 ml of saline. Each exúdate was passed through clean gauze to remove particulate matter, and the leukocytes were counted with a hemocytometer. Purity was assessed using modified Wright's staining (Leuko-Stat, Fisher, Orangeburg, NY). Approximately 3 x 107 cells were obtained from each exúdate, and 91% ± 1% of the cells isolated were PMN. Serial dilutions of isolated PMN (1 x 104 to 1 x 106 cells) were then assayed for MPO activity. PMN-enriched brain homogenates were made by adding 2 X 104,5 x 104,andl X 105 isolated PMN to 1 ml aliquots of left hemisphere homogenates from control rats. Standard curves of MPO activity were generated for both PMN isolates and PMN-enriched brain homogenates.
To serve
MPO
Activity Assay
MPO activity was measured in brain tissue homogenates from normal rats and in rats 24 h after trauma or sham insult. MPO activity was also determined in isolated PMN and PMN-enriched brain from normal rats. 365
BIAGAS ET AL. Isolated PMN and brain tissue pellets were resuspended in 2.5 ml of hexadecyltrimethylammonium bromide (HTAB, 0.5% HTAB in 50 mM potassium phosphate buffer, pH 6.0, Sigma, St. Louis, MO). HTAB is a detergent that releases the MPO enzyme from leukocyte granules. Three freeze-thaw cycles were performed to further disrupt granules. Samples were centrifuged for 10 min at 2500g for a final time. Supernatant (100 p,l) was added to 2.9 ml of 50 mM potassium phosphate buffer with o-dianisidine hydrochloride (0.167 mg/ml, Sigma, St. Louis, MO) and H202 (0.0005%, Sigma, St. Louis, MO). Change in absorbance at 460 nm was assayed spectrophotometrically (Beckman, Somerset, NJ) for 2 min in triplicate. MPO activity was calculated as the mean of the three readings. One unit of MPO activity was defined as the degradation of 1 p.mole of H202/min (Barmen, 1969). Brain tissue results were expressed as units of MPO activity per gram of tissue. An estimate of accumulated PMN per gram of tissue was made from extrapolated MPO activity in PMN-enriched brains.
Statistical Analysis All data are presented as mean ± SEM. Brain tissue MPO activity in normal rats represents mean MPO activity of right and left hemispheric sections. Between-group comparisons of MPO activity in normal (n 12), sham (n 5), trauma (n 12), and PMN-depleted-trauma (n 12) rats were made 24 h posttrauma using one-way ANOVA and the Student-Newman-Keuls test. MPO activity in isolated PMN and in PMN-enriched brain tissue was analyzed by linear regression. Hématologie variables measured before and 24 h after trauma were analyzed in both trauma (n 6) and PMN-depleted-trauma (n 12) groups using Student's of /-test. paired Between-group comparisons hématologie variables were made using one-way ANOVA and Student-Newman-Keuls test. A p value less than 0.05 was considered to be statistically =
=
=
=
=
=
significant. RESULTS All rats in all groups survived. As previously described in this model, rats recover, are extubated within 1 h, and are returned to their cages shortly thereafter (Schoettle et al., 1990). Physiologic variables, including arterial blood gases, mean arterial blood pressure, and rectal temperature, were measured in all rats at the time of surgery. There was no difference in any physiologic variable. Rats that were PMN-depleted before trauma differed from trauma rats in body weight, but all rats were within the predefined weight range of 350-450 g (430 ± 11 g vs 379 ± 6 g, PMN-depleted-trauma rats vs trauma rats, respectively, p < 0.05). Hématologie variables were measured in serial blood samples in a total of 18 rats ( 12 with PMN-depletion) before and 24 h after trauma (Table 1). An increase in total white blood cell count was observed 24 h posttrauma. This was due to increases in both absolute PMN and monocyte counts. No such increase in total white blood cell count was seen in vinblastine-treated rats. Vinblastine treatment markedly reduced the circulating absolute PMN count before trauma and 24 h posttrauma but did not decrease circulating monocyte or lymphocyte counts. Posttraumatic monocytosis occurred in nondepleted rats. In vinblastine-treated rats, the absolute monocyte count tripled, although this increase was not statistically significant. Hematocrit was slightly reduced by vinblastine treatment before and 24 h after trauma (p < 0.05) (Table 1). The reduction in hematocrit was 7% and should be expected to have minimal, if any, physiologic significance. A single 0.5 mg/kg dose of vinblastine had no effect on the circulating platelet count, as previously reported in our model (Uhl et al., 1992). MPO activity measured in isolated homologous PMN was linearly correlated to the number of PMN present (r2 0.99, p < 0.001)(Fig. 1). Brain tissue contains inhibitors of MPO activity that are mostly but perhaps not completely eliminated in the modified MPO assay (Barone et al., 1991). To equate the MPO signal to number of PMN in the brain, MPO activity was determined in PMN-enriched brains (Fig. 2). MPO activity was detected in all PMN-enriched brain aliquots. PMN count and MPO activity in PMN-enriched brain were linearly correlated (r2 0.96, p < 0.01) (Fig. 2). Tissue MPO activity in normal brain was 0.090 ± 0.022 U/g and between 0.060 U/g and 0.125 U/g in sham. In our study, MPO activity in normal rats was higher than previously reported in a model of spinal cord trauma using saline-perfused but nonwashed tissue (Xu et al., 1990). However, our results do concur with those of Barone et al. (1991), using a nearly identical technique in rat brain. =
=
366
BRAIN MYELOPEROXIDASE ACTIVITY AFTER TRAUMA Table 1.
Hematologic Variables Measured 24 H After Trauma"
HCTb
T
(%)
V
WBC
t
(109/L)
V
ANC
T
(109/L)
V
AMC
T V T V
(109/L) ALC
(109/L)
Before
trauma
43.7 40.8 7.27 6.54 0.89 0.07 0.05 0.10 6.31 6.37
0.8 0.6** 0.49 0.58 0.29 0.03* 0.03 0.02 0.66 0.56
in
Rats Before
After 44.5 39.5 9.53 6.68 2.64 0.27 0.33 0.31 6.51 6.05
and
trauma
± ± ±
± ± ±
± ± ± ±
1.3 0.6** 0.43* 0.61 0.42* 0.10** 0.08* 0.10 0.64 0.57
"Rats received intravenous vinblastine sulfate or saline vehicle 5 days before Circulating hematocrit and leukocyte counts before and 24 h after trauma are shown. bT, trauma (n 6); V, vinblastine administered PMN-depleted-trauma (n 12); Hct, hematocrit; WBC, complete white blood count; ANC, absolute polymorphonuclear leukocyte count; AMC, absolute monocyte count; ALC, absolute lymphocyte count. *p < 0.05 before trauma vs after trauma. **p < 0.05 vinblastine administered vs trauma. trauma.
=
=
MPO activity in the right hemisphere of the trauma group was different from MPO activity in either hemisphere of all other groups tested (p < 0.05) (Fig. 3). A fivefold increase in MPO activity was observed in sections from the injured hemisphere 24 h after trauma (0.448 ±0.13 U/g vs 0.090 ± 0.022 U/g in trauma vs normal, respectively, p < 0.0'j) (Fig. 3). No increase in MPO activity was observed in sections from the contralateral, uninjured hemisphere in trauma rats or sections from either hemisphere in sham rats. Rats depleted of PMN before trauma showed no increase in brain MPO activity in either the injured or contralateral hemisphere 24 h after trauma.
DISCUSSION
Supporting our primary hypothesis, we demonstrated a fivefold increase in brain tissue MPO activity 24 h after percussive trauma in rats. This information corroborates and augments previous work in this model using
0.90
o
1
0.40 -0.3O
-o
>
-
5
0.00
Control
R
L
R
L
R
L
Myeloperoxidase (MPO) activity (U/g) in normal (mean ± SEM of both hemispheres, Dn= 12), sham (craniotomy only,S«-= 5), injured (right) and uninjured (left) hemispheres in rats with trauma ( 0« 12), and rats with polymorphonuclear leukocyte (PMN)-depletion and trauma ( n 12). A fivefold increase in MPO activity is demonstrated in injured hemisphere of trauma rats. No increase in MPO activity is seen in either uninjured hemisphere or in injured hemisphere of rats rendered PMN-depleted before trauma. PMND, PMN-depleted. *p < 0.05 vs all other FIG. 3.
=
=
groups.
368
BRAIN MYELOPEROXIDASE ACTIVITY AFTER TRAUMA
increase in brain tissue MPO activity, suggests that the increase in brain tissue MPO 24 h posttrauma specifically represents PMN infiltration. It also suggests that a 93% reduction in the circulating absolute PMN count at the time of trauma prevents accumulation of PMN in brain tissue 24 h posttrauma. Using these observations and data extrapolated from the relation between PMN count and MPO activity in PMN-enriched brain tissue, we estimated PMN accumulation in traumatized tissue. A fivefold increase in MPO activity represents an accumulation of approximately 5 X 106 PMN/g brain tissue (about 1.5 X 106 PMN in each 300 mg section of injured brain tissue sampled in each rat) in the traumatized hemisphere during the first 24 h posttrauma. This study did not rule out a contribution of tissue macrophages (microglia) to the observed increase in brain MPO activity. As with monocytes, however, significant increases in the number of reactive microglia are not evident until 24 h or more posttrauma (Giulian et al., 1989). Furthermore, MPO activity in saline-perfused brain tissue from control rats, which would be expected to reflect tissue macrophages, was low but detectable. That MPO activity in the left hemisphere of vinblastine-treated rats was similar to control values suggests that a small level of non-PMN-generated MPO activity is present in brain tissue. Additional studies in rats not subjected to trauma would be needed to further address the source of this MPO activity. The mechanism(s) and location(s) of PMN accumulation were not addressed specifically in this study. However, saline perfusion of tissue should have eliminated free PMN in the vascular space, excluding them from MPO measurement. The remaining PMN are likely to be from several sources: (1) PMN infiltrating tissue, (2) PMN adhering to vascular endothelium, and (3) PMN in microthrombi and hemorrhage. These are all PMN that have pathologic location and play a potential role in local parenchymal and vascular injury. Such conclusions are supported by our previous work, in which PMN accumulation 4-8 h posttrauma exceeded changes in brain blood volume (Schoettle et al., 1990). If PMN or other leukocyte cell lines play important pathophysiologic roles in posttraumatic brain injury, recent advances in the ability to modify PMN accumulation and function by means of specific antibodies to adhesion receptors on both PMN and vascular endothelium may lead to significant improvements in therapy (Clark et al., 1991). In conclusion, a fivefold increase in brain PMN during the first 24 h after percussive trauma was demonstrated through the combined use of a recently modified MPO assay and selective PMN depletion produced by vinblastine sulfate in rats. This MPO assay can be used to assess the effects of various therapeutic interventions on posttraumatic PMN accumulation in the brain. In addition, depletion of circulating PMN attenuated posttraumatic PMN accumulation in the brain. Moreover, as effective PMN-endothelial-adhesion receptor antagonists are not readily available for the rat, PMN depletion with vinblastine is a potentially useful tool for the initial evaluation of the role of PMN in posttraumatic brain pathophysiology.
ACKNOWLEDGMENTS We acknowledge the generous support of a grant from the Brain Trauma Foundation and the American Heart Association, Pennsylvania Affiliate. We thank Drs. Frank Barone and Len Hillegass from SmithKline Beechman Research Laboratories and Dr. Heidi Horner from Athena Neurosciences for helpful input on the MPO assay. We thank Dr. Shekhar Venkataraman for critical review, Lisa Cohn for editorial assistance, and Janet Santucci, Carleen Heinz, and Becky Gmuer for preparation of this manuscript.
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