Journal of Cerebral Blood Flow and Metabolism 17:647-658 © 1997 The International Society of Cerebral Blood Flow and Metabolism Published by Lippincott-Raven Publishers, Philadelphia

Hypoxia-Ischemia, But Not Hypoxia Alone, Induces the

Expression of Heme Oxygenase-l (HSP32)

in Newborn Rat Brain

*tMarcelle Bergeron, tDonna M, Ferriero, :j:Hendrik J. Vreman, :j:David K. Stevenson, and *tFrank R. Sharp

Department of Neurology, *Veterans Affairs Medical Center and fUniversity of California, San Francisco, California; and

:j:Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA

Summary: Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme to produce bile pigments and carbon monoxide, The HO-I isozyme is induced by a variety of agents such as heat, heme, and hydrogen peroxide, Evidence suggests that the bile pigments serve as antioxidants in cells with com­promised defense mechanisms, Because hypoxia-ischemia (HI) increases the level of oxygen free radicals, the induction of HO-I expression in the brain during ischemia could modulate the response to�oxidative stress, To study the possible involve­ment of HO- l in neonatal hypoxia-induced ischemic tolerance, we examined the brains of newborn rat pups exposed to 8% O2 (for 2,5 to 3 hours), and the brain of chronically hypoxic rat pups with congenital cardiac defects (Wi star Kyoto; WKYI NCr), Heme oxygenase-l immunostaining did not change after either acute or chronic hypoxia, suggesting that HO- l is not a good candidate for explaining hypoxia preconditioning in new-

Hypoxia induces the expression of a set of stress pro­

teins called oxygen-regulated proteins, which have been

implicated in the development of drug and radiation re­

sistance in tumor cells (Heacock and Sutherland, 1990).

Among these proteins, heme oxygenase-l (HO-I; also

called HSP32) has received increasing attention (Maines,

1992). Heme oxygenase catalyzes the degradation of

Received September 1 2, 1 996; final revision received January 6, 1997; accepted January 24, 1 997.

This work was supported by National Institutes of Health grants nos. NS28 1 67 and NS 1 4543 (F.R.S . ) , P20NS32553 (D.M.F.) , and HD-14426; The Hess Research Fund, and The Mary L. Johnson Research Fund (D.K.F). M.B . was a recipient of a postdoctoral fellowship from The Medical Research Council of Canada.

Address correspondence and reprint requests to: Dr. Marcelle Bergeron, Department of Neurology (V 1 27), UCSF and V A Medical Center, 4 1 50 Clement Street, San Francisco, CA 94 1 2 1

Abbreviations used: BSA, bovine serum albumin; CO, carbon mon­oxide; HI, hypoxia-ischemia; HO, heme oxygenase; HSF, heat shock factor; P7, postnatal day 7; PB , sodium phosphate buffer; WKY/NCr, Wistar Kyoto rats with congenital heart diseases, normotensive, and chronically hypoxic.

647

born rat brain. To study the role of HO- l in neonatal HI, l -week-old rats were subjected to right carotid coagulation and exposure to 8% 02/92% N2 for 2.5 hours. Whereas HO enzy­matic activity was unchanged in ipsilateral cortex and subcor­tical regions compared with the contralateral hemisphere or control brains, immunocytochemistry and Western blot analy­sis showed increased HO-l staining in ipsilateral cortex, hip­pocampus, and striatum at 12 to 24 hours up to 7 days after HI. Double fluorescence immunostaining showed that HO-l was expressed mostly in ED-l positive macrophages. Because ac­tivated brain macrophages have been associated with the re­lease of several cytotoxic molecules, the presence of HO-l positive brain macrophages may determine the tissue vulner­ability after HI injury. Key Words: Heme oxygenase­HSP32-Hypoxia-Ischemia-Macrophage-Neonatal brain-Tolerance.

heme molecules derived from hemoproteins such as cy­

tochrome P-450, nitric oxide synthase, tryptophan pyr­

rolase, and several peroxidases and catalases, to biliver­

din, ferrous iron, and carbon monoxide (CO). Biliverdin

is then converted to bilirubin by biliverdin reductase

(Maines, 1992). Two different gene products that both

contribute to HO enzymatic activity (microsomal iso­

zymes HO-l and HO-2) have been characterized (Maines et aI., 1986; Shibahara et aI., 1993). Whereas

HO-2 activity is refractory to most types of stress or

injury, HO-l is induced by various stimuli. In healthy

unstressed adult rat brain, most of HO activity has been

attributed to the HO-2 isozyme whereas the HO-l iso­

zyme seems to be present only at very low levels (Sun et aI.,

1990; Ewing and Maines, 1991, 1992; Maines, 1992).

The 5'-flanking promoter region of HO-l gene con­

tains several important regulatory elements including at

least one copy of the following: a heat shock element (Shibahara et aI., 1987; Okinaga and Shibahara, 1993), a site for nuclear factor-kappa B (NFkB) (Lavrosky et

648 M. BERGERON ET AL.

aI., 1994), an AP-1-Iike binding site (MUlier et aI., 1987;

Alam and Zhining, 1992), and a metal regulatory ele­ment (MUller et aI., 1987) that may modulate the re­

sponses to heat shock or denatured proteins, hypoxia or

oxidative stress, Fos/Jun immediate early genes, and

heme, respectively. Indeed, after global and focal isch­emia (Paschen et aI., 1994; Takeda et aI., 1994; Nimura

et aI., 1996), hyperthermia (Ewing and Maines, 199 1)

and subarachnoid administration of lysed blood or he­

moglobin (Matz et aI., 1996; Turner et aI., 1997) in adult

rats, as well as heat or hydrogen peroxide exposure in

primary cultures of neurons and astrocyteS (Dwyer et aI.,

1995), the induction of HO-l messenger RNA and pro­

tein is observed mainly in nonneuronal cells. Increased

HO-l messenger RNA and protein have also been re­ported in rat brain after glutathione depletion (Ewing and

Maines, 1993). There is evidence to suggest that the

bilirubin produced by HO activity may serve as an anti­oxidant in cells with compromised defense mechanisms

(Stocker et aI., 1987). This may be important for neurons

which have low levels of the common antioxidants glu­

tathione and ascorbate (Raps et aI., 1989). Because hyp­

oxia and ischemia have been associated with increased

free oxygen species (Siesjo et aI., 1989), the production

of antioxidants by HO-l could help protect the brain from oxidativ,e injury.

Hypoxia pretreatment has been shown to confer neu­

roprotection against hypoxic-ischemic (HI) injury in

newborn rats (Gidday et aI., 1994). Though the molecu­

lar mechanism underlying this hypoxic preconditioning

is still poorly understood, HO-l could play a role be­

cause it is an oxygen-regulated protein. Indeed, protec­

tion against subsequent lethal insults has been shown

after prior induction of HO-l either by UV A exposure in

skin fibroblasts (Vile et aI., 1994), hemoglobin pretreat­

ment in rat (Otterbein et aI., 1995), or overexpression in

rabbit coronary microvessel endothelial cells (Abraham

et aI., 1995). To elucidate the role of HO-l in neonatal

hypoxia and ischemia, the present study describes the

effect of acute and chronic hypoxia and the effect of

ischemia on HO-l expression in newborn rat brain. In

view of the suggested protective effects of hypoxia pre­

conditioning against ischemic brain damage in the neo­

natal rat (Gidday et aI., 1994), we also investigated the

role of oxygen-regulated HO-l protein as a potential can­

didate responsible for neonatal hypoxia-induced isch­

emic tolerance.

MATERIALS AND METHODS

Animal preparation All procedures were perfonned in accordance with the Na­

tional Institutes of Health Guide for Care and Use of Labora­tory Animals, and all protocols were approved by the Univer­sity of California at San Francisco Committee on Animal Re­search. Male and female Sprague-Dawley rats (Bantam Kingman, Fremont, CA, U.S.A.) were used. Seven litters each

J Cereb Blood Flow Metab. Vol. 17. No.6. 1997

containing 10 Sprague-Dawley pups at postnatal day 7 (P7), 4 litters each containing 7 to 10 normotensive chronically hyp­oxic Wi star Kyoto pups at P7 (WKY/NCr) bearing various combinations of cardiac anomalies including hypertrophic car­diomyopathy, defects of the aortic arch system, and Tetralogy of Fallot (Kuribayashi et a!., 1990) and, 3 litters of 10 P7 Wi star pups as the control strain were used as previously described (Rice et a!., 1981; Ferriero et aI., 1990). Rat pups at P7 were used because their brain maturity is grossly comparable to that of a late-term gestation human fetus or newborn infant (Dob­bing and Sands, 1979). After anesthesia with a gas mixture containing 1 % halothane in 70% N20 and 30% O2, rat pups underwent the right common carotid artery coagulation through a ventral midline neck incision. The wound was sutured and pups returned to their dam for 2 hours. Pups were then placed in an 8% 02/92% N2 humidified atmosphere in a chamber partially submerged in a water bath maintained at a constant temperature of 37°C (HI group; coagulation, hypoxia). Sham­operated animals underwent the same operative procedure ex­cept that the carotid artery was not ligated (hypoxia-treated group; no coagulation, hypoxia). Control untreated group (no coagulation, no hypoxia) animals and a group of coagulation­only animals (with no hypoxia; n = 5) were also studied. Because the latter two groups showed no difference in cell integrity and HO-l expression, the control animals shown in the present study refer to the untreated control group (no co­agulation, no hypoxia). In general, animals from each litter were divided into control (1 to 2 per litter), hypoxia-treated (2 to 3 per litter), and HI groups (6 to 7 per litter). The systemic oxygen saturation in live P7 animals (untreated WKY/NCr and normal Wi star) was determined with an oxygen transducer probe (Oxisensor II N-25, Nellcor Inc., Hayward, CA, U.S.A.) wrapped around the pups abdomen and connected to a Nellcor Pulse Oximeter.

Western blot analysis At 24 hours after hypoxia or HI, rats were deeply anesthe­

tized with an intraperitoneal injection (0.3 g/kg) of Nembutal (pentobarbital sodium; Abbott Lab, North Chicago, IL, U.S.A.) and killed by decapitation. Brains were quickly removed and dissected on ice. The cerebral cortex, hippocampus, and stria­tum from each hemisphere were placed in Laemmli solubiliz­ing buffer (2.5% sodium dodecyl sulfate, 10% glycerol, 62.5 mmol/L Tris-HCI, pH 6.8, 5% 2-mercaptoethanol) and boiled for 10 minutes. The whole tissue extract was then frozen at -70°C. Western immunoblot analysis was performed as de­scribed previously (Bergeron et aI., 1996) with modifications. Protein concentration was determined using the bicinchoninic acid method (Pierce, Rockford, II, U.S.A.). Equal amounts (55 fLg) of protein per sample were separated on 12% sodium do­decyl sulfate polyacrylamide gels with 4.5% stacking gel. After electrotransfer onto a nitrocellulose membrane (0.2 fLm; Schlei­cher and SchueH, Keene, NH, U.S.A.), immobilized proteins were stained with Ponceau solution to verify equal protein loading. After a brief rinse in deionized water, the membranes were incubated overnight at 4°C in 0.1 moUL sodium phos­phate buffer (PB) pH 7.4, containing 5% nonfat dry milk, 1% bovine serum albumin (BSA) and 0.1 % Tween-20, rinsed briefly in 0.1 mollL PB containing 1 % BSA and 0.1 % Tween-20, then incubated for 2 hours with a I :3500 dilution of rabbit polyclonal anti-rat HO-I antibody (StressGen, Victoria, BC, Canada). This polyclonal antibody, raised against rat liver pu­rified HO-l protein, was originally described by Maines et aI., (1986). After three washes, membranes were incubated with a 1 :2500 dilution of anti-rabbit Ig-horseradish peroxidase anti­body (Amersham, Arlington Heights, IL, U.S.A.) for 1.5 to 2

HO-I EXPRESSION IN ISCHEMIC NEWBORN RAT BRAIN 649

hours. Finally, the membranes were washed three times and the bound antibody was visualized with the ECL chemilumines­cence system according to the manufacturer's protocol (Amer­sham). A computer-based imaging system (MCID, Imaging Research, St-Catherines, Ontario, Canada) was used to measure the areas of HO-I protein on Western immunoblot autoradio­grams. The relative density of HO-l protein (32 kDa) bands was analyzed after subtraction of the film background.

Immunocytochemistry At the appropriate times after each treatment, rats were anes­

thetized with -Nembutal and perfused through the left ventricle with cold 4% paraformaldehyde made up in 0.1 moUL PB, pH 7.4. Brains were removed from the skulls, postfixed in 4% paraformaldehyde for 1 to 4 hours and stored in a 30% sucrose overnight at 4°C. Fifty micrometer-thick coronal sections were cut on a vibratome and washed twice with 0.05 moUL PB. After 1 hour incubation in a peroxidase-inhibiting solution (0.65% sodium azide and 0.2% hydrogen peroxide in 0.05 moUL PB, pH 7.4), sections were incubated for 2 hours in a blocking solution (5% nonfat dry milk, 2% goat serum, 1 % BSA, 0.1 % Triton X-IOO, and 0.1 % rat serum, made up in 0.1 moUL PB, pH 7.4). The sections were then incubated for 12 to 48 hours at 4°C with the same anti-rat HO- l antibody used for Western blotting (StressGen, Victoria, BC, Canada) diluted I :4000 in 2% goat serum, 1 % BSA, 0.1 % Triton X-IOO, made up in 0.1 mollL PB, pH 7.4. Alternate sections from each brain were incubated without primary antibody (as negative controls). Af­ter three 10 minute-washes in 0.05 mollL PB, sections were incubated at room temperature for 2 hours with a I :200 dilution of biotinylatep goat anti-rabbit IgG antibody (Vector Labora­tories, Burlingame, CA, U.S.A.). Sections were then incubated in an avidin-horseradish peroxidase solution (Elite Vectastain, Vector Laboratories) for 2 hours, followed by threc washes with PB. Staining was visualized with 0.015% diaminobenzi­dine (Sigma, St-Louis, MO, U.S.A.) and 0.001 % hydrogen per­oxide. Sections were then washed, mounted on gelatin-coated slides and coverslipped.

Double immunofluorescence labeling To identify which cell type stained for HO-I, some sections

were coincubated with rabbit polyclonal anti-rat HO-l antibody (1 :4000) and either the mouse monoclonal anti-rat antibody ED-l (uncharacterized cytoplasmic antigen expressed by all cells of the rat monocyte/macrophage lineage; Serotec, Oxford, U.K.; 1:2000), OX-42 (complement type 3 receptor found on both resting and activated monocytes, macrophages, neutro­phils, and microglia; Serotec, Oxford, U.K.; I :4000). or glial fibrillary acidic protein (GFAP) present in astrocytes (ICN, Costa Mesa, CA, U.S.A; 1 :4000). All dilutions were performed in 2% goat serum, 1 % BSA. 0.1 % Triton X-IOO, made up in 0.1 mollL PB, pH 7.4. After 24 to 48 hours incubation at 4°C, sections were washed three times for 10 minutes in PB and incubated for 2 hours in the dark with a Texas-Red-conjugated, goat anti-rabbit IgG antibody (I : 150; Vector Laboratories) to­gether with a biotinylated goat anti-mouse IgG antibody (1: 150; Vector Laboratories). After three 10 minute washes, labeled sections were incubated for 2 hours with avidin­conjugated fluorescein isothiocynate (FITC; Vector Laborato­ries; 1: 150 made up in a solution containing 0.1 moUL sodium bicarbonate and 0.15 mollL sodium chloride, pH 8.2). Sections were mounted onto slides and immediately coverslipped with Fluoromount-G (Southern Biotech. Assoc. Inc., Birmingham, AL, U.S.A.). Sections were photographed on a Leitz vario­orthomat microscope using a Ploemopak 2.1 fluorescence illu­minator. The same area of representative sections was photo-

graphed with interchangeable filters for Texas-Red and FITC fluorescence.

Histopathological evaluation Histopathological scoring of each newborn rat brain was

performed blindly on alternate coronal sections stained with cresyl violet (Nissl). Because increased OX-42 and GFAP staining occurs in areas of neuronal loss and brain damage after HI (Sheldon et a!., 1996), alternate sections stained with OX-42 and GF AP antibodies were also examined. The scoring scale was as follows: 0, no injury or no detectable neuronal loss; l, minimal neuronal loss with occasional gliosis; 2, columnar damage in cortex involving predominantly layers II through IV, moderate cell loss with areas of infarction and concomitant gliosis; and 3, severe cell loss and gliosis associated with ex­tensive tissue infarction.

Determination of heme oxygenase enzymatic

activity Twenty-four hours after HI, rat pups were anesthetized with

Nembutal and decapitated. Brains were removed and dissected on ice. The cerebral cortex and a subcortical region comprised of hippocampal, striatal, thalamic, and hypothalamic tissue were isolated from each hemisphere. To account for the CO that may be bound to erythrocytes in the cerebral blood vessels, we perfused some animals with cold saline (0.9% sodium chlo­ride) before processing the tissue and found no difference in HO activity compared with nonperfused animals. The tissue was weighted and homogenized by sonication (4 pulses of 1 second; Branson Sonifier Cell Disrupter 185 with microtip) in 4 volumes of ice-cold 0.1 moUL potassium phosphate buffer, pH 7.4. The protein concentration was determined using the bicinchoninic acid method (Pierce, Rockford, IL, U.S.A.). Heme oxygenase activity was determined using a gas chro­matographic method as described previously (Vreman and Stevenson, 1988) with some modifications. Briefly, 20 f.1L of brain homogenate was reacted with 20 f.1L of 4.5 mmollL NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) and 20 f.1L of 150 f.1mollL heme in a septum-sealed, amber-colored vial at 37°C in the dark. For blank values, the NADPH solution was replaced by an equal volume of potas­sium phosphate buffer. For negative controls, I f.1L of 600 f.1mollL chromium mesoporphyrin (potent inhibitor of HO ac­tivity) was added to the complete reaction mixture (Vreman et a!., 1993). This procedure inhibited the HO-induced CO pro­duction down to the blank value, suggesting that the entire CO production in the reaction vials was derived from HO activity. As a positive control, rat pup brain HO activity was induced by a subarachnoid (intracisternal) injection of purified hemoglo­bin, which has been shown to markedly increase HO-I expres­sion in adult rat brain (Turner et a!., 1997). Twenty-four hours after injection, HO activity values in these brain homogenates ranged from 1.02 to 1.33 nmol CO produced/h/mg protein which is about two- to threefold higher than the activity mea­sured in normal newborn brain (Table I). After 5 minutes of preincubation at 37°C in the dark, vials were purged with CO­free air and allowed to incubate for an additional 15 minutes. The reaction was stopped by addition of 2 f.1L of sulfosalicylic acid solution (60% weight-to-volume ratio) and by cooling in wet ice. The amount of CO generated by the enzyme in the vial headspace was analyzed by gas chromatography (Vreman and Stevenson, 1988). Carbon monoxide concentration in tissue extracts was calculated from the peak area of the sample com­pared with that of CO external standards. The chromatographic assay was linear in the range of CO values obtained with brain tissue homogenates.

J Cereb Blood Flow Metab, Vol. 17, No.6, 1997

650 M. BERGERON ET AL.

TABLE 1. HO activity in homogenates of contralateral and

ipsilateral cerebral cortex and subcortical regions of P7 newborn rats 24 hours after hypoxia-ischemia

Control (n = 2) Contralateral (n = 7) Ipsilateral (n = 7)

Cerebral cortex

0.43 ± 0. 1 0 0.43 ± 0.06 0.47 ± 0.08

Subcortical regions

0.56 ± 0.08 0.48 ± 0.07 0.5 1 ± 0.06

Values represent the mean ± SD of triplicate determinations from "n" animals in each group and are expressed in nanomole of carbon monoxide produ'ced/h/mg protein. Intergroup comparisons (between controls and the contralateral and ipsilateral hemispheres) determined by analysis of variance showed no significant difference.

Statistical analysis Histopathological scores are reported as median score val­

ues. Other data represent the mean ± SD expressed as percent of normal (oxygen saturation data) and nanomole of CO pro­duced/h/mg protein (HO activity data). Intergroup comparisons for HO activity data were performed by one-way analysis of variance. All other data were analyzed by unpaired two-tailed Mann-Whitney non parametric test.

RESULTS

Heme oxygenase-! expression in untreated controls

Because there was no difference in the intensity and the profile of HO-l expression between the brain of un­treated controis and the cerebral hemisphere contralateral

to the carotid occlusion (Fig. I, Lanes 1-3 and 4-6,

respectively), only the results obtained from the contra­

lateral hemisphere were included in Figs. 2 to 4. In un­

treated P7 rats, constitutive HO-I immunoreactivity was

detectable throughout the brain (Figs. 2C,E 3A,B, and

4A) with more intense staining in areas of myelinogen­

esis of the white matter such as the cingulum, corpus callosum, internal and external capsule, and around the

periventricular ependyma (not shown). Double t1uores­cence staining of normal P7 rat white matter showed that

almost all HO-l positive cells were ED-I positive mac­

rophages (Fig. 5E,F) and occasionally OX-42 positive

50.0 -

34.9 -

28.7 -

20.9

1

. ,

2 3 4 5

,-

microglia (not shown). In addition to the white matter

staining, constitutive HO-l expression was also found in

the endothelium throughout the brain, in glia-like cells

and some neurons in the cortex (Fig. 4A) and in portions

of the hippocampus such as the hilus of the dentate gy­rus, the stratum oriens, the pyramidal cell layer and the

stratum lucidum of the CA I to CA3 subfields of Am­

mons horn (Figs. 2E and 3B).

Heme oxygenase-! expression after acute and

chronic hypoxia

Acute exposure to 8% O2 (for 3 hours) I day before

ischemia has been shown to confer tolerance to the new­

born Sprague Dawley rat pup brain against ischemic in­

jury (Gidday et aI., 1994). Using the same experimental

protocol, we studied the possible role of HO-I in the

phenomenon of hypoxia-induced ischemic tolerance by

examining brains from rat pups 24 hours after exposure

to 8% O2 for 3 hours. Western immunoblot analysis of

crude brain extracts (Fig. I) showed that the rabbit anti­

rat HO-I antibody used in the present study recognized a

characteristic -32 kDa band that is consistent with the rat

HO-I protein (HSP32) described previously (Maines et

aI., 1986; Ewing and Maines, 1991). In all regions in­

vestigated (striatum, hippocampus, and cortex), there

was no significant difference between untreated controls

(Fig. I, lanes I to 3), the contralateral side that received

the hypoxia without the coagulation (in animals sub­

jected to HI) (Fig. I, lanes 4 to 6), or sham-operated

animals with hypoxia only (not shown). In agreement

with these observations, immunocytochemistry experi­

ments showed no detectable change in HO-I expression

in newborn brain 24 hours after hypoxia (Figs. 2C,E and

4A), compared with untreated control animals (not

shown). Heme oxygenase-I expression in pup brain was

similar to controls whether animals had been exposed to 8% O2 for 2.5 hours up to 3.5 hours or whether brain

tissue was analyzed 0, I, 5, 24, 48, or 72 hours or 4 days

after hypoxia (not shown).

6 7 8 9

- - - ....c HO-1

, .

FIG. 1. Western immunoblot analysis of rat brain heme oxygenase-1 (HO-1 ) protein levels, 24 hours after a hypoxic-ischemic insult induced at postnatal day 7 (P7). Whole tissue extracts of striatum (Lanes 1 , 4, and 7), hippocampus (Lanes 2, 5, and 8), and cerebral cortex (Lanes 3, 6, and 9) were prepared as described in Materials and Methods and equal protein samples (55 I-1g) were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using rabbit anti-rat HO-1 antibody. Lanes 1 to 3: untreated control rat. Lanes 4 to 6: contralateral hemisphere exposed to hypoxia (8%02 for 2.5 hours) without the common carotid artery coagulation. Lanes 7 to 9: ipSilateral hemisphere with both the common carotid artery coagulation and exposure to hypoxia (8%02 for 2.5 hours). Molecular weight markers in kilodalton are indicated on the left. This experiment was repeated four times with similar results. HO-1, heme oxygenase-1 .

J Cereb Blood Flow Metab. Vol. 17, No.6. 1997

HO-I EXPRESSION IN ISCHEMIC NEWBORN RAT BRAIN 651

A B

FIG. 2. Effect of hypoxia and hypoxia-ischemia on brain HO-1 expression in newborn Sprague Dawley rat. Twenty-four hours after the hypoxic-ischemic insult induced at P7, increased HO-1 immunostaining was observed throughout the cortex (0) and in the hilus of the dentate gyrus of the hippocampus (arrows in F) ipsilateral to the common carotid artery ligation. Note the columnar pattern of HO-1 expression in this moderately injured (damage score = 2) ipsilateral cortex (arrows in 0). Contralateral cortex (e) and hippocampus (E) subjected to hypoxia alone showed no increased HO-1 staining compared with normal untreated newborn rat brain. Alternate brain sections incubated without HO-1 primary antibody showed no endogenous staining in corresponding areas of cortex (A) and hippocam­pus (8). Magnification: 10x, scale bar = 200 [.1m.

Induction of HO-l has been reported after chronic in vitro hypoxia in tumor cells (Heacock and Sutherland,

1990). Rat pups (P7) generally survive a maximum of 3.5 to 4 hours in 8% O2 before dying from cardiac arrest

related to severe hypotension and hypoglycemia (Rice et

aI., 1981; Vannucci and Yager, 1992). In the present

study, because brain HO-I expression was not induced

by 3 hours of in vivo hypoxia at 8% O2 , we examined the effect of chronic hypoxia on brain HO-l expression by

studying rats with congenital cardiac defects (WKY /

NCr; normotensive, chronically hypoxic Wistar rats).

The rats of this inbred strain spontaneously develop vari­

ous heart anomalies including Tetralogy of Fallot, ven­

tricular septal defect, pulmonary valve stenosis and/or

hypertrophic cardiomyopathy in association with hypo­

plasia of the ductus arteriosus, and occasional anomalies

in the aortic arch systems (Kuribayashi et aI., 1990).

Because the WKY!NCr rats are from a Wi star rat lin­eage, we used normal Wistar rats as untreated controls.

The percentage of oxygen saturation in live untreated

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652 M. BERGERON ET AL.

FIG. 3. Effect of hypoxia and hypoxia-ischemia on brain HO-1 expression in chronically hypoxic rat pups bearing congenital cardiac defects (Wistar Kyoto, normotensive; WKY/NCr). Normoxic Wistar rats at P7 were used as untreated controls. The pattern of constitutive HO-1 expression found in Wistar rat cortex (A) and hippocampus (8) was similar to that of the Sprague Dawley pup shown in Fig. 2C and 2E respectively. Untreated WKY/NCr rats showed a pattern of HO-1 expression similar to that of Wistar rats except for an increased number of HO-1 immunoreactive neurons in the stratum oriens and pyramidal cell layer of CA1 to CA3 subfields of Ammons horn and in the hilus of dentate gyrus (DG) (arrows in panel 0). Twenty-four hours after hypoxia-ischemia induced in P7 WKY/NCr pups, increased HO-1 immunostaining was observed throughout the severely injured (damage score = 3) ipsilateral hemisphere including the infarcted cortex (E) and hippocampus (F). Contralateral cortex (C) and hippocampus (0) subjected to hypoxia alone showed no change in the pattern of HO-1 expression compared with untreated WKY/NCr and Wistar controls. Magnification: 10x, scale bar = 200 iJm.

WKY /NCr and normal Wi star P7 rat pups was 82.1 ±

7.5% and 95.0 ± 3.0%, respectively (values represent the

mean ± SD of n = 10 animals; P = 0.0015: significantly

different by unpaired two-tailed Mann-Whitney nonpara­

metric test).

The distribution and intensity of HO-l expression in

untreated control P7 Wistar pups was similar to that de­

scribed above for the normal P7 Sprague Dawley rats

J Cereb Blood Flow Metab. Vol. 17, No.6. 1997

(compare Figs. 2C with 3A and, 2E with 3B). The pattern

of HO-l expression in the brain of chronically hypoxic

P7 WKY/NCr rats was similar to that of untreated P7

Wi star controls. In general, chronic hypoxia seemed to

have no effect on constitutive HO-l expression through­

out the brain of P7 WKY INCr pups compared with nor­mal Wistar pups. The only difference in WKY/NCr ani­

mals compared with normal Wistar rats (Fig. 3B,D) was

HO-I EXPRESSION IN ISCHEMIC NEWBORN RAT BRAIN 653

FIG. 4. High-power photomicrographs of contralateral (A,C) and ipsilateral (8,0) cortex from the Sprague Dawley rat pup (A,8) and the WKY/NCr pup (C,O) shown in Figs. 2 and 3 respectively. Whereas HO-1 immunoreactive neurons (arrows) and macrophage/microglia­like cells (small arrowheads) were found in areas of moderate brain injury (8; damage score = 2), severe brain injury (0; damage score = 3) associated with extensive tissue infarction was characterized by intense HO-1 immunoreactivity in macrophage/microglia·like cells and in the remaining endothelium. Contralateral cortex (A,C) subjected to hypoxia alone showed no sign of brain damage and no change in HO-1 expression compared with untreated control rat brain. Magnification: 20x, scale bar = 100 IJm.

a higher density of HO-l neuronal staining in the stratum

oriens and the pyramidal cell layer of CAl to CA3 sub­

fields of the hippocampus and in the polymorphic layer

of the dentate gyrus. Exposure of WKY/NCr rats to 8%

O2 for 2.5 to 3.0 hours had no detectable effect on brain HO-l expression compared with untreated WKY/NCr

animals or the contralateral side (nonligated side with

hypoxia only) of WKY/NCr pups subjected to HI pro­

cedures (Fig. 3C,D).

Heme oxygenase-l expression after

hypoxic-ischemic injury

The perinatal HI model produced in P7 rats is charac­

terized by injury in the hemisphere ipsilateral to the ca-

J Cereb Blood Flow Metab, Vol. 17, No. 6, 1997

654 M. BERGERON ET AL.

rotid occlusion (Rice et aI., 1981). Damage ranges from

the loss of a few isolated cells up to gross infarction in

cortex, hippocampus, striatum, thalamus, and white mat­

ter ipsilateral to the ligation. Moderate (damage score =

2) to severe injury (damage score = 3) is more common

than little (damage score = I) or no damage (damage score = 0) (Table 2). Exposure to hypoxia alone (con­

tralateral side) does not produce any evidence of cellular

damage.

Western immunoblot analysis of crude brain extracts

obtained from Sprague-Dawley rats pups 24 hours after

HI showed that HO-l protein expression was increased 5 to lO-fold in ipsilateral striatum, hippocampus, and cor­

tex (Fig. 1, lanes 7 to 9), compared with the contralateral

side (Fig. 1, lanes 4 to 6) or untreated controls (Fig. 1, lanes 1 to 3). In agreement with these observations, in­creased HO-l immunoreactivity was detected as early as

6 to 12 hours after HI in the ipsilateral cortex near the

corpus callosum (not shown). Markedly increased HO-l

immunostaining was observed in cells from cortex, hip­

pocampus, striatum, and thalamus ipsilateral to the ca­

rotid occlusion by 24 hours after HI (Fig. 2D,F). Heme

oxygenase-l immunostaining was still quite prominent at

4 days after HI (Fig. 5C,D), and still detectable in the remaining tissue at 7 days (not shown). Interestingly, the increased Hq-l expression found in ipsilateral cortex

often displayed a columnar pattern (Fig. 2D, arrows).

Sections incubated without HO-l antibody showed no

endogenous staining (Fig. 2A,B). WKY/NCr pups sub­

jected to HI showed a pattern of brain injury and HO-l

expression very similar to that described above for the

Sprague Dawley pups. As shown in Table 2, there was

no significant difference in the median histological dam­

age score between the two groups (P = 0.9812, Mann­

Whitney nonparametric test). Increased HO-l expression was observed throughout ipsilateral ischemic hemisphere

(Fig. 3E,F) compared with the contralateral hypoxic side

TABLE 2. Distribution of histopathologic damage scores in Sprague Dawley and WKYINCr newborn rats 24 hours

afier hypoxia-ischemia

Animals

Damage SPD WKY/NCr score* (n = 19) (n = I I )

0 I I 1 3 2 2 II 5 3 4 3

Median scoret 2 2

SPD, Sprague Dawley; WKY/NCr, Wi star Kyoto rats with congen­ital heart diseases, normotensive. and chronically hypoxic.

* Histopathological scores: 0, no gross or histological damage; I, no gross damage. minimal neuronal loss; 2, columnar cortical infarction, moderate neuronal loss; 3, extensive infarction and gliosis. severe neu­ronal loss.

t p = 0.98 1 2 : the median damage scores for the two groups are not significantly different by Mann-Whitney nonparametric test.

J Cereb Blood Flow Metab, Vol. 17, No.6, 1997

(Fig. 3C,D). For all animals investigated in this study,

the intensity of HO-l staining was generally proportional

to the degree of brain injury. Whereas moderate injury (Fig. 2D,F) resulted in increased HO-l expression in

some neurons and glia-like cells (Fig. 4B), severe injury (Fig. 3E,F) resulting in large areas of brain tissue infarc­

tion revealed an increase in HO-l-positive macrophage­

like cells and sustained HO-l staining in the remaining

endothelium (Fig. 4D). Induction of HO-l protein ex­

pression did not increase total HO enzymatic activity in

ipsilateral cortex and subcortical regions 24 hours after

HI (Table 1).

Double immunofluorescence staining after

hypoxia-ischemia

To define the type of cells expressing HO-l protein,

double fluorescence labeling using antibodies against

HO-I and ED-I, OX-42 or GFAP was performed 24

hours and 4 days after HI in newborn Sprague-Dawley

rats. Heme oxygenase-I expression in ipsilateral cortex

(Fig. 5A to D) was found mainly in brain macrophages

expressing the ED-l antigen. Double immunolabeling

with HO-l and OX-42 or GFAP antibodies showed oc­casional co staining of HO-l/OX-42 or HO-l/GFAP (not

shown). Twenty-four hours after moderate HI injury,

most but not all of the HO-l-positive cells were found to

be ED-l positive (Fig. 5A,B). Almost all HO-l positive

cells were found to be colabeled with the ED-I antigen 4

days after HI (Fig. 5C,D).

DISCUSSION

Hypoxia preconditioning and heme oxygenase-1

expression

Hypoxia pretreatment (8% 02/3 hours) confers neuro­

protection in newborn rats against ischemic injury 24

hours after the initial preconditioning (Gidday et aI.,

1994). Such hypoxia treatment has relatively no effect on

neuronal integrity (Rice et aI., 1981) and on several

physiological parameters such as regional cerebral blood

flow and water content (Vannucci et aI., 1988; Mujsce et

aI., 1990), NADH fluorescence (Welsh et aI., 1982),

brain protein synthesis (Dwyer et aI., 1987), and cerebral

calcium uptake (Stein and Vannucci, 1988). In addition,

the expression of several genes including the immediate

early genes fos and jun (Munell et aI., 1994), HSP72

(Ferriero et aI., 1990; Munell et aI., 1994) and GFAP

(Burtrum and Silverstein, 1994) are unaffected by such

in vivo hypoxia pretreatment in newborn rat brain.

Recent studies have suggested that the induction of

oxygen-regulated proteins could be involved in the

mechanism of hypoxia-induced ischemic tolerance. In­

creased expression of certain proteins including HO-l

has been reported in tumor cell lines (Heacock and Sutherland, 1990), endothelial cells (Zimmerman et aI.,

HO-i EXPRESSiON iN ISCHEMIC NEWBORN RAT BRAIN 655

FIG. 5. Double fluorescence labeling of cerebral cortex sections from newborn Sprague-Dawley rats 24 hours and 4 days after hypoxic­ischemic injury. Brain coronal sections (50 fJm) were stained with HO-1 antibody (Texas Red, red) and ED-1 antibody (FITC, green/yellow) as described in Materials and Methods. Panels A, C and E are from the same corresponding field as panels B, 0 and F respectively. Co-expression of HO-1 and ED-1 was observed in ipsilateral cerebral cortex from newborn rats 24 hours (A,B) and 4 days (C,D) after a hypoxic-ischemic insult induced at P7. Whereas not all HO-1-positive cells were colabeled with ED-1 at 24 hours after injury (compare A and B), almost all HO-1-positive cells were found to be colabeled with ED-1 after 4 days (compare C and D). Panels E and F show the double labeling of a control P7 rat cingulum. Nearly all HO-1-positive cells (E) observed in the cingulum were also colabeled intensely with ED-1 (F). Magnification: 40x, scale bar = 50 fJm.

1991), and astrocyte cultures (Kuwabara et aI., 1996)

exposed to hypoxia followed by reoxygenation. A role

for HO-l as a candidate protein responsible for hypoxia

preconditioning was suggested because prior induction

of HO-l protein protects against subsequent lethal insults in several biological systems (Vile et aI., 1994; Abraham et aI., 1995; Otterbein et a!., 1995; Vogt et aI., \995).

Previous in vitro studies have shown that hypoxia stimu­

lated the DNA binding activity of the transcription fac­

tors NFkB (Koong et aI., 1994) and heat shock factor

(HSF; Benjamin et aI., 1990). Although the promoter

region of HO-l gene contains a NFkB binding site (Lavrosky et aI., 1994) and a heat shock element (Shiba­

hara et aI., 1987; Okinaga and Shibahara, 1993), the

J Cereb Blood Flow Metah, Va!. 17. No.6. 1997

656 M. BERGERON ET AL.

present study failed to show an induction of HO-l pro­

tein expression after the 2.5 to 3.5 hour-hypoxia treat­

ment necessary for preconditioning and neuroprotection.

The HO-l induction observed in cultured cells may be

caused by low oxygen tensions for long periods of time that cannot be achieved in vivo. For this reason, we in­vestigated the effect of chronic in vivo hypoxia on brain

HO-l expression in rat pups with congenital heart de­

fects (WKY/NCr). These animals also failed to show

increased HO-l protein expression. In contrast to the

study of Gidday et aI., (1994) which used acute hypoxia

(8% O2 for 3 hours) to induce ischemic 'tolerance, the

present study showed that chronic neonatal hypoxia as­sociated with congenital heart defects (WKY/NCr rats) did not protect the brain against subsequent HI injury

(Table 2). Taken together, these observations suggest that the induction of HO-l expression is not the mecha­

nism responsible for hypoxia preconditioning in new­

born rat brain.

Effect of hypoxia-ischemia on heme oxygenase-!

expression

Perinatal HI brain damage is associated with increased HO-l protein expression. The degree of HO-l expression in the hemisphere ipsilateral to the common carotid ar­

tery occlusioq was dependent on the severity of the in­sult. After moderate injury, HO-l expression was in­

creased mostly in brain macrophages found throughout

the focal areas of tissue damage and in scattered neurons,

some astrocytes, and endothelial cells ipsilateral to the

carotid occlusion. Severe insults resulting in extensive

tissue infarction were accompanied by HO-l staining in

ED-I-positive macrophages and the surviving endothe­

lium almost exclusively. Heme oxygenase-I-positive as­trocytes were infrequent despite massive and progressive

reactive astrogliosis in areas of HI brain damage in the

newborn (Burtrum and Silverstein, 1994; Sheldon et aI.,

1996). In contrast, HO-IIED-l positive macrophages in­

creased in number and became the major cellular com­

ponent expressing HO-l after HI. The distribution of

these HO-l-positive cells was found to be similar to that

recently reported for ED-I-positive cells in the same animal model of neonatal HI (Ivacko et aI., 1996). The

origin of these HO-l-positive macrophages is not

known. Some of the HO-IIED-l macrophages observed

in the present study may have entered the brain from blood vessels possibly in response to the breakdown of

blood-brain barrier (Vannucci et aI., 1993) and signals

from damaged neurons and glia. Alternatively, some of the HO-IIED-l macrophages might have been derived

from brain-resident macrophages and possibly microglia

(Thomas, 1992). Glutathione depletion (Ewing and Maines, 1993) as

well as hyperthermia (Ewing and Maines, 1991) in rats

significantly induce HO-l messenger RNA and protein

J Cereb Blood Flow Metab. Vol. 17. No.6. 1997

without concomitant increased brain HO activity. Simi­

larly, the present study showed that despite a 5- to 10-

fold increase in HO-l expression after HI, HO activity

remained unchanged compared with controls. Heme oxy­

genase-2 protein, which is found mainly in neurons (Ew­

ing and Maines, 1992; Maines, 1992), is the most abun­dant HO isozyme in the brain and is responsible for the

bulk of brain HO activity (Sun et aI., 1990; Ewing and

Maines, 1991). With the ongoing neuronal loss associ­

ated with HI injury, it is possible that the increased HO-l

expression observed mainly in proliferating macro­

phages may compensate for the loss of neuronal HO-2

protein and thus, maintain the overall HO activity at a

normal leveL Although it is also possible that HO-l pro­tein becomes inactive after HI, the lack of detectable changes in brain HO activity after neonatal HI does not rule out the possibility for a local effect of HO-l activity

resulting in the release of free iron, bile pigments,and CO.

Carbon monoxide (like nitric oxide) has been pro­

posed as a putative neurotransmitter acting as a physi­

ologic regulator of guanylyl cyclase and cGMP­

dependent protein kinase (Maines, 1993; Verma et aI.,

1993). Recent studies have shown that the activation of metabotropic receptors could modulate neuronal HO ac­

tivity and in tum, CO could be involved in the signal transduction pathway coupling these receptors to the ac­

tivity of the NaK-ATPase pump (Glaum and Miller,

1993; Nathanson et aI., 1995). In view of this suggested

relationship between glutamate receptor activation and

increased HO activity in the brain, it is possible that the

increased HO-l expression observed in neurons after moderate HI injury may be caused by, at least in part, the

overactivation of glutamate receptors (Rothman and Ol­

ney, 1986). Ischemia-induced oxidative stress and cellu­lar protein denaturation may also induce neuronal HO-l

through activation of specific transcription factors such

as fos and jun, NFkB, and HSF. Indeed, a region­

selective induction of the immediate early genes fos and

jun has been reported ipsilateral to the HI injury in new­

born rat brain (Munell et aI., 1994). Moreover, increased

of DNA-binding activity at the AP-l binding site and the

heat shock element has been reported in cerebral cortex

after transient focal ischemia in adult rats (Salminen et

aI., 1995) and in gerbil hippocampus after global isch­

emia (Nowak and Abe, 1994). However, because neu­

rons only express HSF-2, which is less able than HSF-l

to direct a strong heat shock response (Marcuccilli et aI.,

1996), a major increase of HO-l expression in neurons

through HSF activation seems less likely. Interestingly,

increased NFkB binding activity was noted in ischemic

cortex only 5 days after focal ischemia (Salminen et aI.,

1995), suggesting that this late NFkB surge may be related

to postischemic infiltration of inflammatory macrophages.

Increased production of the antioxidant bilirubin

(Stocker et aI., 1987) after induction of HO-l expression

HO-J EXPRESSiON IN ISCHEMIC NEWBORN RAT BRAIN 657

could protect neurons from subsequent delayed injury.

However, this study showed that although there was a

clear attempt by neurons to synthesize more HO-l pro­

tein after moderate injury, there was also a clear failure

for them to survive more severe HI insults. These obser­vations are consistent with previous reports suggesting

that when ischemia is sufficiently severe to produce in­

farction, transcription and/or translation of the heat shock

genes is blocked in neurons and glia destined to die,

whereas the surviving endothelial cells, which have been

shown to induce both HSP72 and HO-l after focal isch­

emia in adult rat (Nimura et aI., 1996) or neonatal HI

(Ferriero et aI., 1990; present study), continue to synthe­

size stress proteins (Gonzalez et aI., 1989; Kinouchi et aI., 1993). In addition, studies have shown that neurons

in culture have a very limited capacity to induce HO-I

expression even after hydrogen peroxide exposure. In

fact, this may contribute to their selective vulnerability to

oxidative stress (Dwyer et aI., 1995). The protective

mechanism suggested for HO-l activity may also in­

volve the potential lethal effects of ferrous iron, which if

un sequestered, may exacerbate HI brain injury by in­

creasing the formation of hydroxyl radicals through the

Fenton reaction (Braughler et aI., 1986). Almost two

thirds of the iron in the brain is stored as ferritin (H and L isoforms). l-I-ferritin (heavy-chain) is found mainly in

neurons and has a low storage capacity consistent with

the high iron utilization. L-ferritin (light-chain) which is

localized mainly in brain macrophages is involved in

long-term iron storage, consistent with the major role of

this cell type as a scavenger (Connor et aI., 1994). As a

result of HI, the increased pro-oxidant levels produced

by HO-l activity combined with the low iron-storage

capacity of neurons (H-ferritin) could contribute to HI neuronal injury. In contrast, because of the greater iron­

sequestering capacity of L-ferritin, brain macrophages

may be able to survive greater increases in iron release

and thus, may better benefit from the antioxidant activity

of HO-I induced expression. Because inhibition of

phagocytic and secretory functions in mononuclear

phagocytes after ischemia has been shown to reduce

ischemic injury in the spinal cord (Giulian and Robert­

son, 1990), it is suggested that HO-I-protected newborn

macrophages, besides scavenging iron and removing cel­

lular debris from developing and/or injured brain tissue,

may also contribute to neuronal damage after HI and

reoxygenation by releasing neurotoxic molecules (Giu­

han et aI., 1993).

Though the bilirubin produced by HO-l may act as an

antioxidant in many tissues, it may also be toxic to the

brain. Hyperbilirubinemia is commonly observed during

the first week of life in humans and rats (Maines, 1992).

Whereas physiological hyperbilirubinemia alone does not cause bilirubin encephalophathy (kernicterus), cer­

tain conditions such as asphyxia and acidosis can predis-

pose the brain to bilirubin toxicity by decreasing biliru­

bin binding to serum albumin and increasing tissue bind­

ing of bilirubin (Maines, 1992). Besides its suggested

role as an antioxidant at moderate concentrations (Stocker et aI., 1987), bilirubin has also been shown to be toxic to cultured astrocytes, neurons, and neural cell lines

(Amit and Brenner, 1993). Thus, the elevated levels of

bilirubin produced by local increases in HO-l expression

after HI injury could contribute to neuronal damage after

ischemia and to the neuronal injury in kernicterus after

perinatal asphyxia.

Acknowledgment: The authors thank Ronald J. Wang for expert technical assistance with the heme oxygenase activity assay.

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