Transient induction of heme oxygenase after cortical stab wound injury

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 38 (1996) 251-259

Research report

Transient induction of heme oxygenase after cortical stab wound injury Bamey E. Dwyer

a,c,d, *,

Robert N. Nishimura b,c, Shi-Yi Lu

a,c,

Alex Alcaraz

a

a Molecular Neurobiology Laboratory, The Department of Veterans Affairs Medical Center, Sepulveda, CA 91343, USA b In Vitro Remyelination Laboratory, The Department of Veterans Affairs Medical Center, Sepulveda, CA 91343, USA c Department of Neurology, UCLA School of Medicine, Los Angeles, CA 90024, USA d Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024, USA Accepted 5 December 1995

Abstract

Heme oxygenase (HO) exists as two isoenzymes designated heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). HO-1 has been identified as a heat shock or stress protein and is inducible whereas HO-2 is largely refractory to induction. HO-2 is the predominant isoenzyme in normal brain and appears to have a predominantly neuronal distribution in cerebral cortex. Cortical stab wound injury resulted in HO-1 induction as determined by Western blot analysis. Immunohistochemical analysis suggested that induced HO-1 was largely restricted to reactive astrocytes and macrophage-like cells. Enhanced HO-1 immunoreactivity was observed in hypertrophied, GFAP + reactive astrocytes near the wound margin as early as 12 h after injury. Very rarely were HO-1 + neurons observed and then only up to 6 h after stabbing. Maximal numbers of HO-1 + astrocytes were found 3 days after stabbing. Their numbers declined thereafter. By 5 days after stab injury few HO-1 ÷ reactive astrocytes were observed although GFAP-- reactive astrocytes were still prominent near the wound margin. HO-1 + macrophage-like cells were initially observed between l and 3 days after injury and they persisted in the margin of the wound for at least 14 days. The proximity of HO-1 ÷ cells to the wound margin suggests that factors associated with injury contribute to the regulation of HO-1 in injured cortex. Keywords: Heme oxygenase; Brain injury; Reactive astrocyte; Heat shock protein; Stress protein; Gliosis

1. Introduction Heme oxygenase (HO) catalyzes the rate-limiting step in heme degradation yielding biliverdin which is subsequently reduced enzymatically to bilirubin [24,50-52]. HO is present in several mammalian tissues including brain and has been resolved into two components designated heme oxygenase-1 and herne oxygenase-2 (HO-1 and HO2, respectively) [24]. HO-1 is a heat shock protein [40] which is inducible by a variety of stresses including exposure to heavy metals, oxidizing stress, ultraviolet radiation, and supraphysiological temperature [6,8,17,18,29,30,42,47,57]. HO-2, which is the major heine oxygenase isozyme in normal CNS [9,11,53], appears largely refractory to induction except, possibly, by gluco-

Abbreviations: HO-1, heme oxygenase-1 isozyme; HO-2, heme oxygenase-2 isozyme; HO, total heme oxygenase; GFAP, glial fibrillary acidic protein. * Corresponding author. Research Service (151), VA Medical and Regional Office Center, North Hartland Road, White River Junction, VT 05009, USA. Fax: (1) (802) 296-6308. 0169-328X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 9 - 3 2 8 X ( 9 5 ) 0 0 3 4 1 -X

corticoids [55]. HO enzymatic activity in brain is among the highest measured in several organs and comparable to that of the spleen [24], an observation which suggests it has an important role in normal CNS function. HO likely participates in the normal turnover of heme containing proteins in brain. HO may also contribute to the regulation of heine-containing enzymes, possibly by regulating access to heme or via the action of carbon monoxide. Thus, HO activity is the sole known physiological source of carbon monoxide (CO), a putative diffusible, neural messenger molecule [11,23,25,27]. CO, like the well-studied neuroregulatory molecule, nitric oxide (NO), may regulate cGMP levels through its ability to activate guanylyl cyclase [25,26,54] and contribute to the regulation of physiologic function including synaptic function [15,43,58]. Previous work from this laboratory showed that HO-1 was a stress protein in cultured rat glial cells but not cultured cortical neurons [6,8]. Here we extend our study of heme oxygenase in the CNS to an in vivo model of cortical stab injury in which reactive gliosis is a prominent feature [33]. Our results in vivo parallel those reported in vitro [8]; HO-1 is inducible after cortical stab injury in reactive

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astrocytes near the wound margin whereas there was little evidence for neuronal induction.

2. Materials and methods

ice-cold 4% paraformaldehyde/0.1 M PBS overnight after which it was cut in blocks and placed in 15% sucrose/0.1 M PBS overnight. The brain slabs were frozen in pre-cooled isopentane and stored in Zip-Loc plastic bags at - 7 0 ° C if not used immediately.

2.1. Stab wound model o f traumatic brain injury

2.3. Western blot

The method for stab injury was based on that of Logan et al. [22]. Male Wistar rats weighing 200-210 g (Simonsen Laboratories, Gilroy, CA) which had been fasted overnight were sedated with methoxyflurane and anesthetized with ketamine (48 m g / k g , i.m.) in combination with sodium pentobarbital (16 m g / k g , i.p.). This dose was usually sufficient to render rats unresponsive to tail pinch for at least 60 min. The scalp was shaved and anesthetized rats were placed in a stereotaxic apparatus for surgery. Core temperature was monitored with a rectal probe and maintained at 37°C __+0.5°C with a heating pad during surgery and in the immediate post-surgical period, about 30 min until rats began to move about their cage. The scalp was cleansed with Povidone-iodine solution (Rugby Laboratories, Inc., Rockville Center, NY) and a midline incision was made on the scalp to expose the skull. A stereotaxic drill was sighted 3 mm to the right of the midline and 2 mm posterior to bregma and a hole was drilled with a 1 mm spherical bit. A second hole was made 3 mm to the right of midline and 4 mm posterior to the first hole. The area between the holes was drilled out to produce a 4.5 mm slit over the right cortex without abrading the dura. Q-tips were used before and after the stab wound to remove blood from the surgical field. A knife blade with a 4.5mm edge and a 4.5 mm stop notch was fashioned from a razor blade and attached to the stereotaxic apparatus to ensure a perpendicular drop. The knife edge was inserted into the brain through the slit and immediately removed. The slit was covered with gelfoam (Upjohn, Kalamazoo, MI) and the scalp was sutured. The duration of surgery was about 10 min. Saline was used to keep the eyes moist during surgery. After surgery Povidone-iodine solution was reapplied to the sutured scalp followed by bacitracin. Rats were allowed to survive from 1 h to 2 weeks. This procedure was approved by the ' animal studies subcommittee at the Sepulveda VA Medical Center. 2.2. Tissue fixation

Rats were deeply anesthetized with methoxyflurane, the chest cavity was opened, and a tube was inserted through the left ventricle into the aorta. Rats were perfused with 110 ml of ice-cold saline containing heparin (1000 U / I ) followed by 400-500 ml of ice-cold 4% paraformaldehyde ( w / v ) in 0.1 M sodium phosphate, pH 7.4 by gravity flow over about 30 min. The rats were placed in the refrigerator for at least 4 h. The brain was then removed and placed in

Tissue punches from the site of the cerebral stab wound were removed and homogenized with sample buffer containing 2% SDS and proteins were separated on 12% SDS-polyacrylamide gels as previously described [32]. Proteins were transferred onto Immobilon P transfer membranes (Millipore Corp., Bedford, MA) by applying a current of 0.3 A for 16-18 h in a BioRad transblotting cell. Blots were stained with antibody HO-1713 [8] diluted 1:1000. The blots were washed with Tris-buffered saline (20 mM Tris-HC1, pH 7.6, 137 mM NaC1) and incubated 1 h with secondary antibody. HO-1 immunostaining was detected using ECL (Amersham Corp., Arlington Heights, IL) according to the manufacturer's recommendations. 2.4. Immunohistochemistry

Coronal sections of 20 /zm were taken at 50-100 /zm intervals through the brain in the area of the stab and mounted on glass slides for immunohistochemistry using rabbit anti-rat HO-1 antibody HO-1713 [8], rabbit anti-rat HO-1 (SPA-895, StressGen, Victoria, BC, Canada) or purified rabbit anti-human GFAP (G-9269, Sigma Chemical Co., St. Louis, MO). Tissue sections were washed twice with 0.1 M PBS and endogenous peroxidase was quenched with 1% hydrogen peroxide in methanol. Sections were washed with 0.1 M PBS and incubated for 30 min at room temperature in 5% normal goat serum in 0.1 M PBS. Sections were incubated at 4°C overnight with primary antibody (HO- 1713 diluted 1:800, StressGen antiHO-1 diluted 1:400, or anti-GFAP diluted 1:400) in buffer comprised of 1% bovine serum albumin, 0.3% Triton X-100, 0.1 M PBS. After repeated washings in 0.1 M PBS tissue sections were incubated for 1 h with biotinylated anti-rabbit IgG (ExtrAvidin Peroxidase Staining Kit for rabbit, Sigma) followed by incubation with ExtrAvidinperoxidase according to the manufacturer's protocol. Reddish brown staining was obtained by incubating sections with 0.05 M acetic acid buffer (pH 5.0) containing 0.02% AEC and 0.005% hydrogen peroxide for 5 min at room temperature. 2.5. Double label immunohistochemistry

Sections were incubated with either HO-1713 or antiGFAP antibody and color was developed with the ExtrAvidin Peroxidase kit as described above. After washing with buffer the second antibody was applied and bluish

B.E. Dwyer et al. / Molecular Brain Research 38 (1996) 251-259

gray staining was obtained using the Vector SG Substrate Kit (Vector Laboratories, Burlingame, CA).

3. Results 3.1. Cerebral stab wound

Histological examination of brain sections taken through the stabbed area revealed a defined tissue gap and the presence of red blood cells. Macrophage-like cells near the wound margin were observed to increase between 1 and 3 days after injury. By 14 ,days after injury the wound was characterized by the persistence of macrophage-like cells, fusion of the wound gap in places, and encapsulation of brain surfaces by GFAP ÷ processes suggesting the formation of a glia limitans. 3.2. Heme oxygenase stabbed brain

Twenty-seven stabbed rats and 20 sham-operated controls were studied: 1 h (2,2), 2 h (2,2), 4 h (2,1), 6 h (1,1), 12 h (2,2), 1 day (5,3), 3 clay (6,3), 5 day (3,3), 7 day (1,1) and 14 day (3,2). There was a transient increase in the number of HO-1 ÷ cells which appeared maximal 3 days

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after stab injury (Fig. 1). We suggest that enhanced immunoreactivity in tissue sections represented an induction of HO-1 since two anti-HO-1 antibodies were used to immunostain the sections shown in Fig. 1 and because Western blot analysis showed an increase in immunoreactive HO-1 in cortical tissue from the area of the wound margin (Fig. 2). From 1 to 6 h after injury HO-1 immunostaining was unremarkable except for the occasional detection of HO- 1 + cells which morphologically resembled neurons (unpublished results). HO-1 ÷ cells with the morphology of reactive astrocytes were clearly present in superficial areas of the cortex near the wound margin at 12 h after injury and were more extensive by 24 h. By 3 days after injury these HO- 1 ÷ cells were maximally observed. Under higher magnification the majority of HO-1 ÷ cells appeared to be hypertrophied, reactive astrocytes with numerous thick processes (Fig. 3A,B). These cells did not stain positive with antibody to macrophage-microglial marker CD-11 although macrophage-like cells in the wound margin described below were stained (unpublished results). The distribution of HO-1 ÷ reactive astrocytes was more closely restricted to the area of the wound whereas GFAP ÷ reactive astrocytes were more widespread (Fig. 3D). This was confirmed by double-label immunohistochemistry where

Fig. 1. Herne oxygenase-1 immunoreactivity in stabbed cerebral cortex 3 days after injury. Three arrowheads indicate the wound margin. 2 0 / z m mounted sections were immmunostained with antibody HO-1713 (A) or with StressGen's rabbit anti-rat HO-1 (B) as described in Section 2. Note numerous intensely stained cells near the wound margin (magnification 40 X , bar = 250 /zm).

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Orioin v

m ~

I

the wound area 14 days after injury when HO-1 + astrocytes were rarely observed.

4. Discussion

43kDa 33kDa

1

2

3

4

5

6

Fig. 2. HO-I induction in stabbed cerebral cortex detected by Western blot analysis. Tissue punches were obtained from the brain of a stabbed rat (lanes 1-3) or from a sham-operated control (lanes 4-6). Lane 1: Tissue punch from stabbed cortex 3 days after injury. Lane 2: Tissue punch from contralateral cortex of same rat. Lane 3: Tissue punch from cerebelllum. Lane 4: Tissue punch from operated hemisphere of sham-operated rat. Lane 5: Tissue punch from contralateral hemisphere of same rat. Lane 6: Tissue punch from cerebellum. Note: HO-1 migrates with an approximate molecular size of 33 kDa and is found only after stab injury in the stabbed cortex. A second band designated HO-L (heine oxygenase-like immunoreactivity) [8] which migrates around 40-42 kDa is found in all brain areas sampled. It is as yet unidentified.

GFAP was found to co-localize with HO-1 in numerous reactive astrocytes near the wound margin but GFAP+/HO - 1- reactive astrocytes were clearly evident at greater distances from the wound (Fig. 4A,B). H O - I ÷ / G F A P - cells with macrophage-like morphology appeared at the wound margin between 1 and 3 days after injury and were commonly observed by 5 days after injury (Fig. 4C). Their identity was confirmed using macrophage marker ED1 (Fig. 4E). In contrast, 5 days after stab injury relatively few HO-1 ÷ reactive astrocytes remained. This was not due to the resolution of astrocyte reaction since GFAP ÷ reactive astrocytes were still abundant in injured cortex (Fig. 4C,D). Seven to fourteen days after injury large, GFAP ÷ astrocytes with thick, extensive processes were conspicuous near the wound margin where GFAP + processes appeared to be forming a layer at the wound surface (unpublished results). HO-1 ÷ macrophage-like cells were still present in

Stab wound injury to cerebral cortex produces a hemorrhagic, necrotic lesion, disruption of the blood-brain barrier, infiltration of inflammatory cells, and reactive gliosis [33]. Our first observation was the induction of HO-1 in large, hypertrophied reactive astrocytes located near the margin of a cortical stab wound. The restricted distribution of HO-1 ÷ reactive astrocytes in injured cortex was distinctly different from that of GFAP + astrocytes which were much more widespread. It more closely resembled the distribution of vimentin-- astrocytes which, when observed at 3 days and later, were also .localized near the wound margin (unpublished results). Even more striking was the observation that the number of HO-1 ÷ reactive astrocytes rapidly decreased after 3 days. The transient appearance of HO ÷ reactive astrocytes in vivo is consistent with our observations that HO-1 can be both rapidly induced and, under certain conditions, rapidly degraded in astrocytes in culture [6-8]. Rapid induction of HO-1 in reactive astrocytes near the wound margin strongly suggests the importance of wound related factors in its regulation although the nature of these factors is speculative. Thus, there is evidence that astrocytes can become phagocytic cells [16]; phagocytosis of heme-containing cell debris may induce HO-1 [41] or sensitize astrocytes to oxidative stress as is suggested to occur in vascular endothelial cells [1]. Alternatively, socalled 'alarm' cytokines, including IL-1, IL-6, and TNF-tx or TGF-fl released at wound sites as part of the acute phase response [2] may be a contributing factor. Thus, HO in liver was inducible by IL-1, TNF, and IL-6 [4,39], and TGF-/3 was recently shown to induce HO-1 in human retinal pigment epithelial cells [20]. IL-1, TNF and IL-6 are found in injured brain [13,49,56] and TGF-/3 is also likely to be present [21,22,37]. It is noteworthy that in injured brain the level of one of these cytokines, IL-1, was elevated by day 1, had peaked at day 2, and was dramatically reduced by day 5 [13] roughly paralleling the appearance of HO-1 ÷ astrocytes in the present study. Rapid disappearance of HO-1 ÷ reactive astrocytes may reflect a diminution of inducing agents at the wound

Fig. 3. Heme oxygenase immunostaining in rat cerebral cortex 3 days after stab wound injury. Triple arrowheads indicate wound margin in A, C, and D. The wound margin in B is at the left. A: Brain section immunostained with antibody HO-1713 demonstrating numerous intensely stained HO-1 + cells with the morphology of reactive astrocytes (100 x magnification, bar = 75 /xm). B: Higher magnification of HO-1 + cells suggests that the majority are hypertrophied reactive astrocytes with numerous thick processes (250 X magnification, bar = 40 /xm). Small arrows in A and B indicate typical hypertrophied reactive astrocytes observed in the study. C: Brain section from stabbed rat immunostained with pre-immune serum as a negative control. (100 x magnification, bar = 75 /xm). D: Brain section from stabbed rat immunostained with anti-GFAP anibody (100 X magnification, bar = 75 /xm). Note the relative restriction of HO-1 -- reactive astrocytes to the area near the wound margin compared to the more widespread distribution of GFAP + reactive astrocytes.

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margin as healing progresses. However, our previous observation that HO-1 is a PEST protein [6] and that rapid loss of immunoreactive HO-1 can be induced in cultured astrocytes [6,7] suggests that regulated proteolytic processes also contribute to loss of immunoreactive HO-1 in reactive astrocytes in vivo. Alternatively, we cannot rule out the possibility that the disappearance of HO-1 ÷ reactive astrocytes reflects the death of these cells. The latter possibility is suggested by the observance of 'apoptotic' cells in the wound area (Apo-Tag, Oncor Scientific) although the identity of these 'apoptotic' cells has not been established. Loss of astrocytes near the wound margin would also be consistent with the report of Blaugrund et al. [3] that astrocytes disappeared from the marginal area of an optic nerve crush injury. Macrophage-like cells were first observed at the wound margin between 1 and 3 days after stab injury and were present for at least 14 days. That these cells are HO-1 ÷ is consistent with ongoing phagocytosis of cell debris and possibly a role also in macrophage activation [41,48]. The delayed appearance of macrophage-like cells in the wound area is consistent with the reports of Femaud-Espinosa et al. [12] and Stichel and Muller [44]. This study raises several interesting questions. Of fundamental importance is whether enhanced HO-1 immunoreactivity in the wound area reflects a true increase in heme oxygenase activity. Preliminary data from this laboratory [7] and the work of others [40] suggest that HO-1 induction is accompanied by elevated enzymatic activity. However, unchanged levels of brain HO enzymatic activity were reported after heat shock in spite of relatively large increases in HO-1 m R N A and HO-1 immunoreactive protein [10]. Secondly, does increased astrocytic heme oxygenase activity contribute to the induction a n d / o r maintenance of the reactive astrocyte phenotype? In this regard, bilirubin, the end-product of heme degradation by heme oxygenase, was reported to induce gliosis [34] as was cadmium and other heavy metals [34] which are known inducers o f heme oxygenase. If so, HO-1 ÷ reactive astrocytes near the wound would seemingly constitute a subpopulation since abundant G F A P - / H O - 1 - reactive astrocytes away "from the wound margin were observed and G F A P ÷ reactive astrocytes persisted in stabbed brain close to the wound margin

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when numbers of HO-1 + reactive astrocytes were diminished. A n important question is whether heme oxygenase contributes to the pathogenesis of cell injury. Thus, bilirubin has been variously described as a neurotoxin [5] and a potentially important cellular antioxidant [31,45,46]. Its relevance in vivo as either a toxin or antioxidant is unclear. In addition, numerous physiological effects are being ascribed to carbon monoxide, a putative neuroregulatory molecule generated by heme oxygenase activity [28,36,38] including effects on cerebral blood flow [19]. CO produced in astrocytes near the wound may diffuse locally. Its possible effects on wound healing is unknown. Lastly, HO generates ferrous ion (Fe 2+) which could contribute to oxidative injury [14]. W e believe this factor is of critical importance in brain cell injury of many types. W e propose that Fe 2÷ generated by heme oxygenase activity directly contributes to CNS cell injury and death. Thus metalloporphyrin inhibitors o f heine oxygenase are neuroprotective in a hippocampal slice model of traumatic CA1 injury [35]. W e propose that heme oxygenase induction by local inflammation or oxidative injury (conditions relevant to CNS trauma and ischemia, tissue transplantation, and chronic neurodegenerative disease among others) can participate in a positive feedback loop. Thus injury-mediated heme oxygenase induction promotes elevated Fe 2÷ which in turn promotes further oxidative injury leading to further stimulation of HO activity and ultimately to tissue destruction.

Acknowledgements Supported by the Research Service of the Department o f Veterans Affairs and the United Cerebral Palsy Research and Educational Foundations, Inc. The authors would like to acknowledge the excellent technical assistance of Su-Ting Fu and W a n g Xiao Wei.

References [1] Balla, J., Jacob, H.S., Balla, G., Nath, K., Eaton, J.W. and Vercellotti, G.M., Endothelial-cell heme uptake from heme proteins: Induction of sensitization and desensitization to oxidant damage, Proc. Natl. Acad. Sci. USA, 90 (1993) 9285-9289.

Fig. 4. Heme oxygenase-1 in stabbed cerebral cortex. A and B: Brain sections from stabbed rat 3 days after injury double stained with antibody HO-1713 and anti-GFAP as described in Section 2. Wound margin is indicated in A by triple arrowheads and is at right side in B. Small closed arrows indicate typical HO-1÷ reactive astrocytes stained brown. Small arrowheads indicate typical GFAP+ reactive astrocytes stained blue. Large open arrows indicate typical reactive astrocytes stained both blue and brown suggesting the co-localization of HO- 1 and GFAP in at least some reactive astrocytes close to the wound margin (A: 100 X magnification, bar = 75 /~m; B: 250 × magnification, bar = 40 /zm). C: Brain section of stabbed rat 5 days after injury double stained with antibody HO-1713 and anti-GFAP antibody. Triple arrowhead indicates wound margin. Abundant GFAP+ reactive astrocytes (stained brown) were found in injured cortex. Small arrows indicate typical HO-1 ÷ cells (stained blue) which were largely restricted to the wound margin and had macrophage-like morphology (magnification 100 X, bar = 75 /zm). D: Brain section from stabbed rat 5 days after injury immunostained for GFAP. Wound margin is at the left. GFAP÷ reactive astrocytes often with long processes (stained brown) are prominent at the wound margin 5 days after stab injury. Note the lack of staining by anti-GFAP of macrophage-like cells at the wound margin (250 × magnification, bar = 40/xm). E: Brain section from stabbed rat 5 days after injury immunostained for macrophage marker, ED-1. Wound margin is at the left. Note numerous ED-1+ cells restricted to the wound margin which had macrophage-like morphology (250 x magnification, bar = 40 /zm).

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