Journal of Neuroscience Research 29:336-345 (1991)

Polymorphonuclear Leukocyte Infiltration Into Cerebral Focal Ischemic Tissue: Myeloperoxidase Activity Assay and Histologic Verification F.C. Barone, L.M. Hillegass, W.J. Price, R.F. White, E.V. Lee, G.Z. Feuerstein, H.M. Sarau, R.K. Clark, and D.E. Griswold Departments of Pharmacology (F.C.B., L.M.H., W.J.P., R.F.W., E.V.L., G.Z.F., H.M.S., D.E.G) and Cell Sciences (R.K.C.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania

Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemia: histologic analysis and a modified myeloperoxidase (MPO) activity assay. Twenty-four hours after producing permanent cortical ischemia by occluding and severing the middle cerebral artery of male spontaneously hypertensive rats, contralateral hemiparalysis and sensory-motor deficits were observed due to cerebral infarction of the frontal and parietal cortex. In hematoxylin-and-eosin-stained histologic sections, PMN, predominantly neutrophils, were identified at various stages of diapedesis from deep cerebral and meningeal vessels at the periphery of the infarct, into brain parenchyma. When MPO activity in normal brain tissue was studied initially, it could not be demonstrated in normal tissues extracted from nonwashed homogenates. However, if tissue was homogenized in phosphate buffer (i.e., washed), MPO activity was expressed upon extraction. Utilizing this modifed assay, MPO activity was significantly increased only in the infarcted cortex compared to other normal areas of the brain. This was observed in non-perfused animals and after perfusion with isotonic saline to remove blood constituents from the vasculature prior to brain removal. The increased PMN infiltration and MPO activity were not observed in forebrain tissue of sham-operated control rats. Also, MPO activity was not increased in the ischemic cortex of MCAO rats perfused immediately after middle cerebral artery occlusion, indicating that blood was not trapped in the ischemic area. By using a leukocyte histochemical staining assay, activity of peroxidases was identified within vascular-adhering/ infiltrating PMN in the infarcted cortex 24 hr after focal ischemia. An evaluation of several blood components indicated that increased MPO activity was 0 1991 Wiley-Liss, Inc.

selective for PMN. The observed increase of approximately 0.3 U MPO/g wet weight ischemic tissue vs. nonischemic cerebral tissues probably reflects the increased vascular adherance/infiltration of approximately 600,000 PMN/g wet weight infarcted cortex 24 hr after focal ischemia. This combined biochemical and histological study strongly suggests that PMN adhere within blood vessels and infiltrate into brain tissue injured by focal ischemia and that the associated inflammatory response might contribute to delayed progressive tissue damage in focal stroke. This modified MPO assay is a useful, quantitative index of PMN that can be utilized to elucidate the potential deleterious consequences of neutrophils infiltrating into the central nervous system after cerebral ischemia, trauma, or other pro-inflammatory stimuli. Key words: PMN, MPO activity assay, focal ischemia, middle cerebral artery occlusion, nervous system, inflammation, rat INTRODUCTION Acute inflammation is characterized by the infiltration of polymorphonuclear leukocytes (PMN) associated with the action of chemotactic mediators (Sny-

Received June 14, 1990; revised November 12, 1990; accepted November 28, 1990. Address reprint requests to Frank C. Barone, Ph.D., SmithKline Beecham Pharmaceuticals, plc, Department of Pharmacology, L-521, P.O. Box 1539, King of Prussia, PA 19406-0939. Abbreviations used: H&E, hematoxylin and eosin; HTAB, hexadecyltrimethylammonium bromide; MCAO, middle cerebral artery occlusion; MPO, myeloperoxidase; PMN, polymorphonuclear leukocytes; SHR, spontaneously hypertensive rats; TTC, triphenyltetrazolium chloride.

PMN and MPO in Focal Ischemia

derman and Uhing, 1988). Generally neutrophils will accumulate in tissues in response to chemotactic factors associated with inflammatory processes (O’Flaherty and Ward, 1979) and products of hemostatic activity (Stecker and Sorkin, 1974). This can occur during ischemia in many tissues, and activated PMN under these conditions can release substances noxious to the vascular endothelium (Harlan, 1985). The degree of inflammatory cell infiltration in inflamed tissue has been estimated histologically, by quantification after tissue digestion or by radiolabeling, in skin (Griswold et al., 1987; Katz and Strober, 1978; Wahba et al., 1984), kidney (Hellberg and Kallskog, 1989; Ormrod et al., 1987), splanchnic vascular beds, e.g., intestine (Gusham et al., 1986), and heart (Chatelain et al., 1987; Engler et al., 1986; Smith et al., 1988) tissues. PMN also have been identified by these methods in models of air embolus-induced focal cerebral ischemia (Hallenbeck et al., 1986; Kochanek et al., 1987) and in a model of laser-induced brain injury (Frerichs et al., 1990). Inflammatory cell infiltration also can be quantified by using an assay for myeloperoxidase (MPO), an enzyme found within the azurophilic granules of PMN (Bradley et al., 1982). MPO is a marker enzyme for these inflammatory cells (primarily neutrophil derived). The MPO activity assay has been found to be a reproducible and objective method to reliably estimate neutrophil infiltration and correlates well with other estimations of PMN movement into inflamed tissues (Allan et al., 1985; Bradley et al., 1982; Change et al., 1986; Goldblum et al., 1985; Krawisz et al., 1984; Mullane et al., 1985; Smith et al., 1988). However, this enzyme assay has not previously been used to quantify PMN in the brain, nor is there information regarding PMN infiltration in the rat middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia. The purpose of the present series of experiments was to demonstrate PMN infiltration 24 hr after cerebral focal ischemia produced by MCAO in the rat through the use of histologic techniques and a modified MPO kinetic assay.

MATERIALS AND METHODS Animals Male spontaneously hypertensive rats (SHR; Taconic Farms, Germantown, NY), 250-310 g in weight, were kept with food and water ad libitum at 23°C and 50% humidity for at least 7 days prior to surgery. SHR were utilized because of their increased sensitivity to MCAO, which in this strain produces large and consistent cerebral infarctions (Barone et al., 1990; Ginsberg and Busto, 1989).


MCAO Surgery Animals were anesthetized with sodium pentobarbital (Steris Laboratories, Inc., Phoenix, AZ; 60 mg/kg, i.p.), and surgical procedures were as described previously (Barone et al., 1990, 1991; Clark et al., 1990; Price et al., 1990; White et al., 1990) with body temperature maintained at 37°C by using a heating pad. The head was shaved and coated with povidone-iodine solution, and the rat was placed in a stereotaxic headholder (David Kopf Instruments, Tujunga, CA) with the skull surface exposed. To verify the effects of MCAO on local cortical microvascular perfusion Laser-Doppler Flowmetry was used. To this end, a 2-3-mm-diameter hole was drilled through the skull above the cortical area receiving blood supply from the artery (i.e., centered at AP=O mm and L = 5 mm from the bregma skull landmark). The probe (PF303; Perimed, Stockholm, Sweden) of a LaserDoppler perfusion monitor (Periflux PF3; Perimed) was positioned on the dural surface and local cortical microvascular perfusion was monitored by using a polygraph (Beckman Instruments, Inc., Schiller Park, IL; model R711 or R5 11A) before (5-10 min), during, and after (20-40 min) MCAO. The middle cerebral artery was exposed through an incision made between the orbit and the external auditory canal with dissection/retraction of the temporal muscle and a 2-3 mm craniotomy made just rostra1 to the zygomatic-squamosal skull suture over the artery. The dura over the MCA was disrupted by using a 30-gauge needle. The tip (made into a 2-mm hook) of a platinumirridium wire (0.0045 inch diameter; Medwire, Mt. Vernon, NY) that was mounted on a micromanipulator was placed under the MCA and the artery was pulled away for simultaneous occlusion and transection by electrocoagulation (Force 2 Electrosurgical Generator; Valley Lab, Inc., Boulder, CO) at the level of the inferior cerebral vein. Permanent MCAO was verified by using Laser-Doppler Flowmetry in the ischemic cortex as described above. In sham-operated animals, the dura was opened over the artery but the artery was not occluded. Following MCAO, the temporalis muscle and skin were closed in two layers. Neurological Testing Twenty-four hours after surgery, two separate neurological examinations were performed to determine the severity of deficits due to sham operation or MCAO surgery. Each animal was classified by using the Neurological Grade (i.e., grading of 0 to 3) as described previously (Benderson et al., 1986b) to define the degree of contralateral forelimb paralysis resulting from the focal ischemic damage in the ipsilateral cortex. A grade of 0


Barone et al.

indicates that no observable deficit was observed, 1 indicates that consistent contralateral forelimb flexion was observed when suspended by the tail, 2 indicates that there was reduced resistance to a lateral push toward the paretic, contralateral side, and 3 indicates that circling behavior towards the paretic side was present. Also, we developed a Hindlimb Placement Test that was performed for each rat. In this test, the rat is held facing away from the edge of a table and the contralateral hindlimb is pulled over the edge of the table and extended downward. A normal response, seen in non-surgically treated animals or ipsilateral to the cerebral surgery, is an immediate placement of the hindlimb back onto the table. An abnormal response was identified as an inability to perform the limb placement.

Histology Following the neurological evaluation, animals were sacrificed by an overdose of sodium pentobarbital. Within 2-3 min, brains were removed and the forebrain was prepared for histologic evaluation or for MPO assay. Also, for comparative purposes, animals were perfused transcardially with 200 ml of isotonic saline solution (25°C at a pressure of 100 mmHg) to flush all blood components from the vasculature. For histologic evaluation, six coronal forebrain slices 2 mm thick were made from the level of the olfactory bulbs to the cortical-cerebellar junction, and the forebrain slices were immersed in a 1 % solution of triphenyltetrazolium chloride (TTC; Sigma Chemical Co., St. Louis, MO) in 1.0 M phosphate buffer (pH 7.4) at 37°C for 20-30 min (Benderson et al., 1986a). Stained tissues then were fixed by infiltration in 10%phosphate-buffered formalin (Lerner Ldboratories, Pittsburgh, PA). Within the next 24 hr, the two sides of each TTC-stained sections were photographed in color by using a Polaroid camera and Polacolor Instant Pack Film (Polaroid Corporation, Cambridge, MA). Following fixation, tissues were prepared for paraffin embedding by routine histologic procedures (Luna, 1968). Tissues were dehydrated through graded ethanol concentrations, cleared in xylene, and infiltrated with Paraplast embedding medium (Monoject Scientific, St. Louis, MO). Following embedding, 6-pm sections were cut on an American Optical Reichert model 820 microtome (Reichert Scientific Instruments, Buffalo, NY), stained with hematoxylin and eosin (H&E; Sigma Chemical Co.; Luna, 1968) and were later examined and photographed by using an Olympus BH-2 microscope (Hitech Instruments, Newtown Square, PA).

solution (25°C at a pressure of 100 mmHg) as follows: Brains were rapidly removed following sodium pentobarbital overdose and cut coronally into 2-mm slices. Slices were coated with OCT embedding medium (Miles Laboratories Inc., Elkhart, IN) and snap-frozen in 2methylbutane (Aldrich Chemical Co., Inc., Milwaukee, WI) chilled in liquid nitrogen. Following sectioning on a Reichert Histostat cryostat (Reichert Scientific Instruments), 6-pm sections were air dried and fixed in alcoholic formalin (Sigma Chemical Co.). Peroxidase(s) activity was visualized through the use of hydrogen peroxide as a substrate, combined with the chromogen p-phenylenediamine (leukocyte peroxidase [myeloperoxidase] histochemical demonstration kit #390, Sigma Chemical Co.).

MPO Activity Assay Brain tissue segments. As described above for histology, some of the sham-operated and MCAO rats were anesthetized with pentobarbital and perfused transcardially with 200 ml of isotonic saline solution (25°C at a pressure of 100 mmHg) prior to brain removal in order to flush all blood components from the vasculature and provide for comparative data with non-perfused animals in the MPO activity assay. For the biochemical determination of MPO activity, the forebrain was dissected from the olfactory bulbs and cut at the cortical-cerebellar junction. The hindbrain and cerebellum were discarded. Based on the location and extent of ischemic damage/ infarctions identified in TTC- and H&E-stained forebrain tissue, each forebrain was sectioned into four separate segments as illustrated in Figure 1. A segment of the ipsilateral frontal-parietal cortex was sliced from the hemisphere ipsilateral to surgery and labeled “A. This segment corresponded to the extent of infarction in animals receiving permanent MCAO but was cut similarly for animals receiving sham- or permanent MCAO-surgeries. Another identical segment was sliced from the contralateral cortical hemisphere, was labeled “B ,” and served as the non-surgery control side for comparison. The forebrain also was dissected into two additional segments at the midline which represented the remaining portion of the hemisphere ipsilateral to surgery (labeled “C”) and the remaining portion of the hemisphere contralateral to surgery (labeled “D”). The four forebrain segments then were immediately frozen at - 80°C for later biochemical analysis. In addition to sham and MCAO animals sacrificed at 24 hr post-surgery, another group of MCAO animals was sacrificed and perfused Histochemical Analyses For Peroxidase(s) transcardially with 200 ml of isotonic saline (25°C at a Several brains were prepared for enzyme histo- pressure of 100 mmHg) immediately (less than 5 min) chemical analysis from MCAO rats that had or had not after MCAO surgery. These animals were prepared in been perfused transcardially with 200 ml isotonic saline order to evaluate MPO activity changes following artery ”

PMN and MPO in Focal Ischemia


Fig. 1. Diagramatic illustration of forebrain dissection into four segments. Segments A and B: The cortex area that exhibits infarction (black area identified in many animals previously) 24 hr following MCAO as described by using the present methods, and the non-surgery contralateral control cortex, respectively. The same areas for segments A and B were prepared from sham-operated control animals. Segments C and D: The remaining portions of the hemispheres ipsilateral and contralateral to surgery, respectively.

occlusion in perfused ischemic tissue prior to possible infiltration of PMN. Analysis of blood components. Various blood components were evaluated for MPO activity in order to determine the specificity for PMN and to establish a quantitative relationship for units MPO activity per PMN. Blood cell isolates including platelets, mononuclear leukocytes (i.e., lymphocytes and monocytes),


PMN, and red blood cells were obtained from two male SHR rats. Whole blood was collected by cardiac puncture in syringes containing heparin (Ely Lilly & Co., Indianapolis, IN; approximately 20 units/ml blood) while the animals were anesthetized with sodium pentobarbital. The samples were centrifuged at 15Og for 45 min (25°C). The platelet-rich plasma was collected to determine if platelets or plasma possesses MPO. The leukocyte buffy coat was harvested and isolations of mononuclear leukocytes, PMN, and leukocyte-free red blood cells were isolated by established methods (Boyum, 1968). Briefly, the separation of mononuclear leukocytes from PMN and red blood cells using the buffy coat was accomplished by centrifugation through Ficoll (Histopaque- 1077, Sigma Diagnostics, St. Louis, MO) after which the PMN were isolated from residual red blood cells by dextran sedimentation (Dextran T500, Pharmacia LKB, Uppsala, Sweden). PMN isolates were greater than 9.5% pure and mononuclear leukocyte isolates were 100% pure. Blood cell isolates were prepared in duplicate in preparation for extraction of MPO. Biochemical assay. The method used to quantitate MPO activity from rat forebrain segments was similar to, but modified from, that recently described for whole rat kidney (Hillegass et al., 1990) and heart tissue pellets remaining after the extraction of creatinine kinase (Griswold et al., 1988). Isolated blood components were extracted and assayed for MPO as per Bradley et al. (1982). The modified MPO activity assay for brain tissue was conducted as follows: Tissue segments were thawed on ice and wet weight in grams was measured. Although not necessary for the MPO assay, but in order to share tissue samples for multiple parameters, each segment was homogenized (model PT/lO35 Polytron, Brinkman Instruments, Inc., Westburg, NY; 5-sec burst at medium intensity) in 4 ml 50 mM Tris-HCI, pH 7.4, 4°C. A standardized 1-ml aliquot (25%) was utilized for the MPO assay. Twenty milliliters 5 mM phosphate buffer, pH 6.0, 4"C, was added to the 1-ml samples and they then were homogenized by using a Tissumizer homogenizer (Tekmar Co., Cincinnati, OH; 3 on/off cycles at 5-sec intervals) and centrifuged at 30,OOOg (30 min, 4°C). The supernatant was discarded and the pellet was washed again as described above. After decanting the supernatant, the pellet was extracted by suspension in 0.5% hexadecyltrimethylammonium bromide (HTAB; Sigma Chemical Co.) in 50 mM potassium phosphate buffer (pH 6.0, 25°C) for approximately 2 min at an original tissue wet weight (adjusted to one-fourth of the tissue) to volume ratio of 1:lO. The sample was immediately frozen on dry ice. Three freeze and thaw cycles then were performed with sonications (10 sec, 25°C) between cycles. After the last sonication, the samples were incubated at 4°C for 20 min and centrifuged at


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12,5008 (15 min, 4°C) in a microfuge (Beckman Instruments, Inc., Spinco Division, Palo Alto, CA). Supernatant MPO activity was assayed as described earlier by Bradley et al. (1982). The rate at which a colored product formed during the MPO-dependent reaction of odianisidine (0.167 mg/ml; Sigma Chemical Co.) and hydrogen peroxide (0.0005%; Sigma Chemical Co.) was measured (i.e., the change in absorbance recorded at 15-sec intervals over a 2-min period) at 460 nm by using a Beckman DU-7 spectrophotometer with kinetic analysis capability (Beckman Instruments, Inc., Irvine, CA). One unit of MPO activity is defined as the amount that degrades 1 (*mole of peroxide/min at 25°C. MPO activity (units) for each forebrain segment was normalized on the basis of grams wet weight tissue.

Statistical Analysis All data are presented as mean 2 SEM. Comparisons between two groups (i.e., the Neurological Grade Test) were made for unpaired data by using Students t-test (Hays, 1973). In the case of non-parametric data (i.e., the Hindlimb Pull Test proportions), the X2 test for two independent samples was utilized (Siegel, 1956). Comparisons between the four different forebrain segment MPO activities were made by using a one-way analysis of variance (ANOVA). Only if the ANOVA was significant was a Dunnet t-test conducted by using the “A” segment for comparison to all other forebrain segments (Winer, 1971). Also, linear regression analysis was carried out for MPO activity expressed in the various blood components (Hays, 1973). Differences were considered statistically significant if P

Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: myeloperoxidase activity assay and histologic verification.

Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemi...
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