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Journal of Neuropathology and Experimental Neurology Vol. 59, No. 3Copyright q 2000 by the American Association of Neuropathologists March, 2000

pp. 218–228

Chronic Prenatal Exposure to Carbon Monoxide Results in a Reduction in TyrosineHydroxylase-Immunoreactivity and an Increase in Choline Acetyltransferase-Immunoreactivity in

the Fetal Medulla: Implications for Sudden Infant Death Syndrome

MARY TOLCOS, BSC (HONS), HUGH MCGREGOR, BSC (HONS), DAVID WALKER, PHD, AND SANDRA REES, PHD

Abstract. Maternal cigarette smoking during pregnancy is associated with a significantly increased risk of Sudden InfantDeath Syndrome (SIDS). This study investigated the effects of prenatal exposure to carbon monoxide (CO), a major componentof cigarette smoke, on the neuroglial and neurochemical development of the medulla in the fetal guinea pig. Pregnant guineapigs were exposed to 200 p.p.m CO for 10 h per day from day 23–25 of gestation (term 5 68 days) until day 61–63, atwhich time fetuses were removed and brains collected for analysis. Using immunohistochemistry and quantitative imageanalysis, examination of the medulla of CO-exposed fetuses revealed a significant decrease in tyrosine hydroxylase-immu-noreactivity (TH-IR) in the nucleus tractus solitarius, dorsal motor nucleus of the vagus (DMV), area postrema, intermediatereticular nucleus, and the ventrolateral medulla (VLM), and a significant increase in choline acetyltransferase-immunoreactivity(ChAT-IR) in the DMV and hypoglossal nucleus compared with controls. There was no difference between groups in im-munoreactivity for the m2 muscarinic acetylcholine receptor, substance P- or met-enkephalin in any of the medullary nucleiexamined, nor was there evidence of reactive astrogliosis. The results show that prenatal exposure to CO affects cholinergicand catecholaminergic pathways in the medulla of the guinea pig fetus, particularly in cardiorespiratory centers, regionsthought to be compromised in SIDS.

Key Words: Brainstem; Catecholamines; Cholinergic; Cardiorespiratory nuclei; Met-enkephalin; SIDS; Substance P.

INTRODUCTION

It is now widely accepted that suboptimal intrauterineconditions account for many of the neurological deficitsthat develop in the postnatal period (1–3). Although thetype, severity, and timing of the insult in relation to ges-tational age are all likely to be important variables, rel-atively little is known of the outcome of specific prenatalinsults. Maternal cigarette smoking during pregnancy isassociated with low birth weight (4) and a significantlyincreased risk of Sudden Infant Death Syndrome (SIDS)(5); it is thought that neural development might be com-promised in infants dying of SIDS (6). Although severalsubtle structural and neurochemical abnormalities havebeen reported in SIDS brains, particularly in the brain-stem (7–12), it is still not certain which specific adverseprenatal conditions might result in these deficits.

Carbon monoxide (CO), one of the agents primarilyresponsible for the adverse effects of cigarette smoke,readily crosses the placenta where it binds to hemoglobincausing fetal hypoxia. Despite the importance of under-standing how CO exposure might affect fetal brain de-velopment, few studies have been undertaken in experi-mental animals. To date it has been demonstrated thatthere are alterations in noradrenaline and serotonin levels

From the Department of Anatomy and Cell Biology (MT, SR), TheUniversity of Melbourne, Parkville, Victoria, and the Department ofPhysiology (HM, DW), Monash University, Clayton, Victoria, Austra-lia.

Correspondence to: Assoc. Prof. Sandra Rees, Department of Anat-omy and Cell Biology University of Melbourne, Parkville, 3052, Vic-toria, Australia.

Source of support: National SIDS Foundation of Australia (Grant #:G93-09/156).

in the brain following chronic prenatal CO exposure (13,14) and in GABA (15) and dopamine (16) followingcombined pre- and postnatal CO exposure. As these werebiochemical studies, the alterations in neurotransmitterlevels could not be localized to specific nuclei. In quali-tative structural studies, gross alterations in the cerebel-lum have been described after combined pre- and post-natal CO exposure (15) and in the cerebral white matter,brainstem, and basal ganglia after acute prenatal exposure(17). However brain development following chronic pre-natal exposure has not been investigated.

Therefore, in this study in the fetal guinea pig, we haveinvestigated the structure and neurochemistry of thebrainstem following chronic prenatal CO exposure duringthe last 60% of gestation. A considerable proportion ofcentral nervous system development in the guinea pigoccurs in utero as it does in the human, making this an-imal a particularly useful model for the human fetus. Wehave concentrated on this region since one of the leadinghypotheses for the cause of SIDS is a dysmaturity orabnormal development of the brainstem nuclei control-ling respiratory, cardiovascular, and arousal activities (6).Reactive astrogliosis has been consistently reported in thebrainstem of SIDS infants (8, 10, 18, 19) and has beeninterpreted as a response to a hypoxic episode. Alter-ations in several neurotransmitter systems in the brain-stem have been reported, including a decrease in the con-centration of the catecholamine synthesizing enzymesphenylethanolamine-N-methyltransferase (PNMT) anddopamine-b-hydroxylase (DbH) (20), an increase in theconcentration of substance P (SP) together with an in-crease in the ratio of SP to met-enkephalin (ME) (11),and a decrease in muscarinic cholinergic receptor binding

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in the arcuate nucleus (12), indicating a disturbance incholinergic transmission.

In the medulla of CO-exposed fetuses near term, wehave quantitatively assessed the immunoreactivity of theneuropeptides substance P (SP), and met-enkephalin(ME); the immunoreactivity of the enzymes involved inthe catecholamine (tyrosine hydroxylase, TH) and ace-tylcholine (choline acetyltransferase, ChAT) synthesisingpathways; the immunoreactivity of the m2 muscarinicacetylcholine receptor (m2), the most abundant musca-rinic receptor type in the brainstem (21); and the prolif-eration of reactive astrocytes using the intermediate fila-ment marker, glial fibrillary acidic protein (GFAP). Anumber of medullary nuclei have been examined, partic-ularly those directly or indirectly involved in the controlof respiratory, cardiovascular, and arousal activities.

MATERIALS AND METHODS

Experimental Procedure

Pregnant guinea pigs (n 5 6) were assigned to either thecontrol (n 5 3) or experimental group (n 5 3) and were placedinto their allocated chamber on days 23–25 of gestation. Guineapigs in the experimental group were exposed to 200 p.p.m COfor 10 h/day until 61–63 days of gestation (term 5 68 days).It has recently been reported that prenatal exposure to 200p.p.m CO throughout pregnancy results in fetal and maternalcarboxyhemoglobin (COHb) levels of 13% and 8.5% respec-tively (22), which is consistent with levels found in humanswho smoke heavily (23, 24). Exposure to CO occurred in aplastic chamber (100 cm 3 50 cm 3 50 cm) sealed with aperspex lid, with food and water provided ad libitum. To gen-erate 200 p.p.m CO, 100% CO (BOC, Australia) was mixedwith air (10 L/min) in a rotameter and delivered to the chamber(22). The concentration of CO in the chamber was monitoredand recorded continuously by a CO sensor built into the sideof the chamber and all gas exited the chamber via an externallyvented passive extraction system. All control animals wereplaced in a separate chamber with the same dimensions as theCO chamber, but were continuously exposed to room air. Theseexperiments were performed under the guidelines set down bythe National Health and Medical Research Council of Australia,and the project was approved by the separate Animal Experi-mentation and Ethics Committees of the University of Mel-bourne and Monash University.

Tissue Preparation

Control and CO-exposed pregnant guinea pigs were removedfrom the chambers at 61–63 days of gestation and fetuses weredelivered by Caesarean section after deeply anesthetizing themothers and fetuses with Nembutal (Pentobarbitone Sodium, 60mg/ml). Fetuses and placenta were weighed and the fetal headperfused via the left ventricle with isotonic saline followed by4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH7.4. Liver weights and crown-to-rump lengths were then mea-sured. Brains were removed from the skull and postfixed for 4h. Whole brain, brainstem (spino-medullary junction to just be-low the cerebellar peduncles) and cerebellar weights were taken

and blocks of brainstem were transferred into 20% sucrose in0.1 M PB (pH 7.4) overnight prior to sectioning.

The medulla of control and CO-exposed fetuses were seriallysectioned at 40 mm on a Leitz-Wetzlar freezing microtome. Ev-ery 5th section was collected for structural analysis. Sectionswere mounted from 0.5% gelatin onto slides coated in 1% gel-atin and stained with 0.01% thionin in acetate buffer (pH 4.4).Following dehydration in graded alcohols, clearing and cover-slipping, sections of medulla from control and CO-exposed fe-tuses were qualitatively assessed for gross structural abnormal-ities such as areas of pallor, necrosis, infarction or neuronalloss, and gliotic aggregations. The intervening sections werecollected and frozen in cryoprotectant (25) until used for im-munohistochemical staining with the antibodies described be-low.

Immunohistochemistry

Immunoreactivity for TH, ChAT, m2, SP, ME, and GFAP waslocalized on free-floating sections using the avidin-biotin per-oxidase complex (Vector Laboratories, Burlingame CA) as de-scribed previously (26). The primary antibodies were obtainedfrom the following sources and used at the following dilutions:mouse-anti-tyrosine hydroxylase (monoclonal, BoehringerMannheim Biochemica, 1:1,000); goat anti-choline acetyltrans-ferase (polyclonal, Chemicon, 1:500); rat anti-m2 muscarinicacetylcholine receptor (monoclonal, Chemicon, 1:1,000); rab-bit-anti-substance P (polyclonal, kindly donated by Dr. R. Mur-phy: the production and characterization of this antibody hasbeen described in Morris et al., 1986 (27), 1:1,000); rabbit-anti-met-enkephalin (polyclonal, Penninsula Laboratories Inc, 1:1,000) and rabbit anti-glial fibrillary acidic protein (polyclonal,Dako, 1:1,000). Sections were incubated either overnight(ChAT, m2, SP, GFAP) or for 48 h (TH, ME). Sections werethen immersed in the appropriate secondary antibody (1:400,biotinylated anti-rabbit IgG; 1:200, biotinylated anti-mouseIgG; 1:200, biotinylated anti-goat IgG; Vector Laboratories) andafter thorough washing placed in the appropriate dilution of theavidin-biotin complex (1:400, SP, ME, GFAP; 1:200, TH; 1:100and Elite kit, m2; 1:50 and Elite kit, ChAT). Sections werereacted with 0.05% 3,39-diaminobenzidine (DAB) solution in0.01% hydrogen peroxidase to produce a brown reaction prod-uct. Phosphate buffer was replaced with Tris phosphate bufferedsaline for ChAT immunostaining to reduce background staining.Sections immunostained with TH, ChAT and m2 were coun-terstained in 0.01% thionin in acetate buffer (pH 4.4) for 6 minand ME was enhanced with 0.01% osmium tetroxide for 30 sprior to dehydration and clearing. Negative controls were pre-pared for each antibody by replacing the primary antibody withPB or primary diluent in the incubation. In this procedure stain-ing always failed to occur. For each antibody, sections fromcontrol and CO-exposed fetuses were stained simultaneously toensure uniform conditions for subsequent quantitative and qual-itative analysis.

Western Blotting

At 62 days of gestation, a control and CO-exposed pregnantguinea pig were removed from the chambers and fetuses (3control and 2 CO-exposed) were delivered by Caesarean section

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after deeply anesthetizing the mothers and fetuses with Nem-butal (Pentobarbitone Sodium, 60 mg/ml). The medulla (spino-medullary junction to just below the cerebellar peduncles) fromeach fetus was collected and homogenized in 50 mM Tris-HCl(pH 7.4) containing 50 mM NaCl and protease inhibitors (10mM EDTA, 10 mM e-amino-n-caproic acid, 0.25 mM PMSF,5 mM N-ethylmaleimide, 10 mM benzamidine, 500 KIU/mlaprotonin and 1 mg/ml pepstatin A). Samples were centrifuged(8 min; 10,000 g) and the supernatant was retained. The con-centration of protein was determined using the bicinchoninicacid (BCA) protein assay reagent kit (Pierce, Rockford, IL) us-ing BSA fraction V (Pierce, Rockford) as standards. Tissuepreparations were then heated (958C for 5 min) in Laemmli’ssample buffer (Sigma Chemical Company, MO, USA) and sep-arated by electrophoresis in 4%–15% gradient Tris-HCl poly-acrylamide gel (BioRad; 40 mg protein/lane) in Laemmli’s run-ning buffer (28). Proteins were electrophoretically transferredonto PVDF nitrocellulose membrane (Immobilon-P transfermembrane, Millipore Corp. Bedford, MA) in 25 mM Tris-gly-cine buffer containing 0.1% SDS and 20% methanol. Mem-branes were then blocked in ‘‘blotto’’ (8% (w/v) skim milkpowder in PS with triton X-100) for 30 min and incubated inmouse-anti-TH (monoclonal, Boehringer Mannheim Biochem-ica, 1:1,000) for 48 h. Membranes were then washed in PB (33 10 min), incubated in biotinylated anti-mouse IgG (1:200;Vector Laboratories) for 45 min and after thorough washing (33 10 min) placed in the avidin-biotin complex (1:200; Vecta-stain Laboratories Burlingame CA) for a further 45 min. Thebands were visualized using DAB as the chromagen and 0.01%hydrogen peroxide. To ensure that the primary anti-TH anti-body was recognizing TH, adult maternal adrenal gland ho-mogenates (prepared as for the medulla) and purified full-lengthTH protein of known molecular weight (0.05 mg and 56 kDa;Signal Transduction Products, San Clemente, CA) were alsoseparated electrophoretically and immunoblotted as described.To control for nonspecific binding of the secondary antibody orthe avidin-biotin complex to proteins, control lanes from whichprimary antibody was omitted were included. In this case, stain-ing failed to occur.

The density of anti-TH-IR bands were measured from controland CO-exposed medullary tissue using the Image Pro Plusimage analysis system as described below. As there were in-sufficient numbers of samples to perform statistics, the resultsare expressed as an average percentage change from controlvalues.

Quantitative Structural Analysis

Every tenth thionin-stained section of medulla extendingfrom the spino-medullary to the ponto-medullary junction wasrandomly projected onto an orthogonal 1 cm 3 1 cm grid. Thecross-sectional area of the medulla was determined using apoint counting technique (see Mallard et al, 1999 (29). In ad-dition the volume of the medulla was determined using theprinciple of Cavalieri (30).

Qualitative Immunohistochemical Analysis

Qualitative assessment of the density and distribution of theimmunoreactivity for TH, ChAT, m2, SP, ME and GFAP in thenuclei of interest was performed double blind by 2 independent

observers. Three levels of the medulla (caudal, mid- and rostral)were chosen (see Figure 3A–C in Tolcos and Rees, 1997 [26])and a section at each level in the control and experimental fe-tuses was analyzed simultaneously using projectorscopes. Theselevels were chosen as they included the relevant areas of car-diorespiratory control, namely the NTS, DMV, hypoglossal nu-cleus, area postrema, ventral respiratory group in the ventro-lateral medulla (VLM) and nucleus ambiguus (NAmb). Othernuclei and tracts were also qualitatively assessed and they in-cluded the spinal trigeminal tract and nucleus (SP, ME, TH),pyramidal tract (GFAP), dorsal and ventral spinocerebellar tract(GFAP), medial longitudinal fasciculus (GFAP), medial lem-niscus (GFAP), inferior olivary (GFAP, ChAT), gracile (TH)and cuneate (TH) nuclei, ventrolateral medulla (SP), ventral re-ticular formation including the intermediate reticular nucleus(TH), hypoglossal, vagus, facial, and abducens nerves (ChAT),as well as the central canal and the lining of the fourth ventricle(GFAP). By analyzing both cardiorespiratory and noncardio-respiratory nuclei we were in a position to evaluate the speci-ficity of any change which might have occurred.

Quantitative Immunohistochemical Analysis

Analysis of immunoreactivity for TH, ChAT, m2, SP, ME andGFAP was performed quantitatively in nuclei in which changeswere observed qualitatively. Sections from the caudal, mid- androstral levels (see Figure 3A–C in Tolcos and Rees, 1997 [26])were assessed quantitatively. The image was projected onto acomputer monitor and a line drawn around the nucleus of in-terest for each section stained with antisera to either TH (NTS,DMV, hypoglossal nucleus, area postrema), ChAT (NTS, DMV,hypoglossal nucleus, facial nucleus), m2 (NTS, DMV, hypo-glossal nucleus, area postrema, facial nucleus), SP (NTS, hy-poglossal nucleus, area postrema), ME (NTS, hypoglossal nu-cleus, area postrema) and GFAP (NTS, DMV, hypoglossalnucleus, area postrema). In the case of TH-, m2- and GFAP-IR, the NTS and DMV were measured together as it was dif-ficult to accurately delineate the nuclei. In addition, the opticaldensity of TH-IR and GFAP-IR was measured in the VLM andthe medullary reticular formation respectively using sample ar-eas of equal size positioned at random. The background opticaldensity was measured from an area where staining was notevident on each section. This was then subtracted from opticaldensity measurements from each nucleus or region. The back-ground-adjusted optical density measurements were averagedfor each animal and then averaged for control and CO-exposedgroups.

Immunoreactivity for all antibodies was assessed quantita-tively using the Image-Pro Plus image analysis system (MediaCybernetics, Maryland). The image was acquired by using aSony (CCD-IRIS/RGB) video camera head mounted to anOlympus microscope. For each objective, the system was cal-ibrated for optical density measurements as follows. An area ofthe slide consisting of the glass slide and coverslip was alignedin the field of view. A black field was obtained by blocking thelight source (infinite optical density), and a blank area of theslide was used as incident light. Using these values, the imageanalysis system converted light intensity into absolute opticaldensity values (range 0 to 2.4). An electronic shading correction

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TABLE 1Data obtained at Postmortem from Control (n 5 8) and

Carbon Monoxide (CO)-exposed (n 5 8) Fetuses

Parameters Control CO-exposed

Litter sizeBody weight (g)Brain weight (g)Cerebellum weight (g)Liver weight (g)

3.00 6 0.6086.64 6 2.26

2.49 6 0.050.29 6 0.013.84 6 0.20

2.70 6 0.7078.35 6 2.59*2.32 6 0.06*0.25 6 0.013.24 6 0.20*

Placental weight (g)Crown-to-rump length (cm)Brain weight: Body

weight (%)Brain weight: Liver

weight (%)

5.40 6 0.6014.00 6 0.40

3 6 0.1

66 6 3

5.99 6 0.2011.94 6 0.40*

3 6 0.1

74 6 6

Values are mean 6 standard error of the mean. * p , 0.05.

was applied to each acquired image to compensate for any un-evenness that might be present in the illumination. Samplingregions of varying stain intensity and a blank region of the slidetested the calibration. A reference slide was used at the startand end of the session to account for any discrepancies.

Counts of Immunoreactive Neurons

In a quantitative study, the numerical density of TH-positiveneurons in the region of the VLM, SP-positive neurons in theNTS and ME-positive neurons in the ventral reticular forma-tion, were determined in sections taken at 3 matched levels incontrol (n 5 8) and CO-exposed (n 5 8) animals. These levelswere caudal, mid- and rostral medulla for TH and mid-, lower,and upper medulla for SP and ME (see Figure 3A–D in Tolcosand Rees, 1997 [26]). It was not possible to count only cellswith a nucleus since the intensity of the immunoreactive prod-uct made it impossible to visualize nuclei therefore all neuronalprofiles were counted. Tyrosine hydroxylase-positive neuronswere counted in the lower quadrant of each section. This areawas demarcated by extending a line from the base of the centralcanal or the fourth ventricle horizontally to the lateral edge ofthe section and another vertically through the raphe nucleus andmedial lemniscus to the ventral edge of the section. This isreferred to as the region VLM in the Results section. The areaof the left and right NTS, and the ventral reticular formationwas determined using adjacent thionin-stained sections. The ar-eas were then measured using a digitizing tablet (SigmaScanPro 4, Jandel Scientific). The results are expressed as cell den-sity (cells/mm2) for each section. The average density of TH-,SP- and ME-positive neurons for each animal was then deter-mined by pooling the results from each level and calculatingthe mean value. For the CO-exposed group, values for eachanimal were corrected for the reduction in the volume of themedulla. Means were then taken for the control and CO-ex-posed groups, and expressed as the mean density (neurons/mm2)6 the standard error of the mean (M 6 SEM). The number ofChAT-IR neurons in the left and right NAmb was determinedby counting the positive neuronal profiles in equivalent sectionsfrom the caudal, mid- and 2 rostral levels in control and CO-exposed animals. An average was determined for the right andleft NAmb, and then for each animal by pooling the results.Means were then taken for control and CO-exposed animalsand the results are expressed as the mean number of ChAT-positive neurons within these levels of the medulla 6 the stan-dard error of the mean (M 6 SEM).

Statistical Analysis

Fetal weights were expressed as the mean 6 standard errorof the mean (M 6 SEM) and analyzed using an unpaired 2-tailed t-test for independent samples assuming unequal vari-ance. All immunohistochemical and structural data is expressedas the mean 6 the standard error of the mean (M 6 SEM) andstatistical analysis performed using the Mann-Whitney U-test.

Preparation of Figures

Black and white negatives (Copex Pan) were scanned ontoan Apple Macintosh computer and figures were compiled usingAdobe Photoshop 4.0. Background artifacts were removed and

image intensity levels were adjusted equally for control andexperimental images.

RESULTS

At 61–63 days of gestation, body, brain, and liverweights were significantly reduced in CO-exposed fetuses(n 5 8) when compared with controls (n 5 8) (Table 1).Measurements of the crown-to-rump length in CO-ex-posed fetuses also showed a significant decrease whencompared with controls. No significant differences werefound in either litter size, cerebellar, or placental weights.There were no significant differences in either brain tobody or brain to liver ratios when CO-exposed fetuseswere compared with controls (Table 1).

Structural Analysis

Analysis of thionin-stained sections of the medulla didnot reveal any gross structural damage or areas of pallor,necrosis, infarction, or gliosis. The latter observation wassupported by optical density measurements for GFAP-IRin the NTS/DMV (0.12 6 0.01 [control] vs 0.11 6 0.01[CO-exposed]) and area postrema (0.34 6 0.03 [control]vs 0.32 6 0.07 [CO-exposed]). Quantitative analysis ofthionin-stained sections of the medulla revealed a signif-icant decrease in the volume (mm3) of the medulla in CO-exposed (82.58 6 3.92) compared with control (92.44 62.81, p , 0.05) fetuses.

Immunohistochemistry

Tyrosine Hydroxylase: In the medulla of control fetus-es, TH-IR neurons processes and terminals were distrib-uted predominantly in the NTS (A2/C2 groups), DMV,hypoglossal nucleus, intermediate and lateral reticular nu-clei, ventrolateral medulla (A1/C1 groups), medullary re-ticular formation, gracile and cuneate nuclei, and spinaltrigeminal nucleus, raphe nucleus, lateral paragigantocel-lular reticular and parvocellular reticular nuclei, and the

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TABLE 2Optical Density Measurements (M 6 SEM) for Tyrosine Hydroxylase (TH) and Choline Acetyltransferase (ChAT)-

immunoreactivity in the Medulla of Control (n 5 8) and Carbon Monoxide (CO)-exposed (n 5 8) Fetal Guinea Pigs

NucleiTH

Control CO-ExposedChAT

Control CO-exposedm2

Control CO-exposed

NTSNTS/DMVDMVXIIAP

—0.15 6 0.03

——

0.37 6 0.02

—0.07 6 0.01*

——

0.21 6 0.06*

0.24 6 0.02—

0.18 6 0.020.27 6 0.01

—

0.28 6 0.02—

0.24 6 0.02*0.33 6 0.02*

—

—0.18 6 0.02

—0.35 6 0.030.75 6 0.06

—0.16 6 0.02

—0.30 6 0.020.69 6 0.1

VLMFacialVLM cells (cells/mm2)NAmb cells

0.10 6 0.01—

10.6 6 0.8—

0.05 6 0.01**—

7.9 6 0.4*—

—0.22 6 0.01

—55.6 6 4.5

—0.22 6 0.01

—53.51 6 4.8

—0.32 6 0.01

——

—0.29 6 0.02

——

* p , 0.05; ** p , 0.01.

area postrema. The distribution is similar to that de-scribed in other species (31–34). The most striking dif-ferences in TH-IR in fibers and terminals in the medullaof CO-exposed fetuses compared with controls was a sig-nificant decrease in the optical density of staining in theNTS/DMV (particularly in the caudal extent of the nu-cleus), AP, and VLM (Table 2). This is illustrated bycomparing Figure 1B (CO-exposed) with Figure 1A (con-trol) and for the area postrema, Figure 1D (CO-exposed)with Figure 1C (control). There also appeared to be adecrease in TH-IR in the intermediate reticular nucleusin CO-exposed (Fig. 1B) fetuses when compared withcontrols (Fig. 1A). The levels of TH-IR were too low togive reproducible quantitative results so optical densitymeasurements were not made. Other regions such as thegracile, cuneate, and spinal trigeminal nuclei did notshow any differences in qualitative analysis. There was asignificant difference in the numerical density of TH-pos-itive neurons in the region of the VLM (refer to Materialsand Methods for description) in CO-exposed fetusescompared with controls (Table 2).

Choline Acetyltransferase: In the medulla of controlfetuses ChAT-IR neurons, fibers and terminals were dis-tributed in the hypoglossal nucleus, DMV, NAmb, med-ullary reticular formation, inferior olivary nucleus, me-dial subnucleus of the NTS, area postrema, lateralreticular nucleus, ventrolateral medulla, cuneate nucleus,lateral paragigantocellular nucleus, spinal trigeminal nu-cleus, facial nucleus, and superior salivatory nucleus.ChAT-IR fibers were also present in the abducens (VI),facial (VII), vagus (X), and hypoglossal (XII) nerves. Thedistribution is similar to that described in other species(35–38). Quantitative densitometry of transverse sectionsof the caudal, mid-, and rostral medulla of control andCO-exposed fetuses revealed a statistically significant in-crease in ChAT-IR within the DMV and the hypoglossalnucleus of CO-exposed animals but not in the NTS (Table2). This is illustrated by comparing Figure 1F (CO-ex-posed) with Figure 1G (control). There was no significant

difference in optical density of the facial nucleus in con-trol compared with CO-exposed fetuses. In a qualitativestudy of the cranial motor nerves (VI, VII, X, XII) andthe inferior olivary nucleus, no consistent differenceswere observed in the intensity ChAT-IR fibers or neurons.No significant differences were found in the number ofChAT-IR neurons of the NAmb in CO-exposed fetusescompared with controls (Table 2).

m2 Muscarinic Acetylcholine Receptor: Within the me-dulla of control fetuses, m2-IR was observed in fibers,terminals, and occasionally over cell bodies in the hypo-glossal nucleus NTS, DMV, ventral and dorsal medullaryreticular formation, lateral reticular nucleus, VLM, NAmb,spinal trigeminal nucleus, gracile and cuneate nuclei, me-dial longitudinal fasciculus, area postrema, arcuate nucle-us, inferior olivary nucleus, raphe nuclei, external cuneatenucleus, gigantocellular, ventral gigantocellular, parvocel-lular, lateral paragigantocellular and intermediate reticularnuclei, Probst’s nucleus and bundle, Roller nucleus, inter-calated nucleus, vestibular nuclei, facial nucleus, preposi-tus hypoglossal nucleus, abducens nucleus, predorsal bun-dle, and medial lemniscus. The distribution is similar tothat described in other species (39–42). There was no sig-nificant difference in the optical density of m2-IR in theNTS/DMV, hypoglossal nucleus, area postrema, or facialnucleus of CO-exposed compared with controls. The re-sults are summarized in Table 2.

Substance P and Met-enkephalin: The distribution ofSP- and ME-IR has been described previously in the me-dulla of the fetal guinea pig (26). Transverse sections ofthe caudal, mid- and rostral medulla were analyzed foralterations in the neurotransmitters/neuromodulators SPand ME. There were no statistically significant differenc-es in optical density in any of the cardio-respiratory nu-clei when control and CO-exposed animals were com-pared. The results are summarized in Table 3. There wasno significant difference between control and CO-ex-posed animals in the density of SP-IR neurons in the

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NTS, or of ME-IR neurons in the ventral medullary re-ticular formation (Table 3). Qualitative analysis revealedno apparent difference in SP- and ME-IR in the spinaltrigeminal tract and nucleus or in SP-IR in the VLM.

Western Analysis

Using quantitative Western analysis, a 13% reductionin the level of TH protein was measured in the medullaof CO exposed fetuses in comparison to controls (C: 0.386 0.03 vs CO: 0.33 6 0.05; Fig. 2).

DISCUSSION

This study has investigated the structural and neuro-chemical development of the fetal brainstem in the guineapig after chronic moderate exposure to CO throughoutthe last 60% of gestation. Significant reductions were ob-served in fetal body, brain and liver weights and crown-to-rump length. There was also a significant reduction inthe volume of the medulla in CO-exposed fetuses com-pared with controls. In contrast to intrauterine growth-restriction produced by chronic placental insufficiency(26), brain sparing was not evident, as there were nosignificant differences in brain to body and brain to liverratios when CO-exposed fetuses were compared withcontrols. In brains from CO-exposed fetuses there wereno gross structural alterations such as necrosis, infarction,or gliosis; the latter was confirmed by optical densitymeasurements of GFAP-IR. To our knowledge this is thefirst study in which the structure of the brainstem hasbeen examined following chronic exposure to moderatelevels of CO. There have been reports of brain damageafter chronic pre- and postnatal exposure to higher levelsof CO (15) and to acute prenatal CO intoxication (17).In this study, the use of quantitative immunohistochem-ical analysis was essential for the identification of region-specific changes in the levels of TH- and ChAT-IR incontrol compared with CO-exposed animals. Althoughthis technique does not measure changes in the amountof protein, the reduction in TH-IR was supported usingWestern blot analysis. The reduction in TH as revealedusing Western blot analysis (13%) was not as great asthat from optical density measurements of single nucleiwithin the medulla (43%–53%). This is not unexpectedas significant changes in specific nuclei are diminishedwhen the entire medulla is homogenized.

A major finding of this study was that prenatal COexposure affected the catecholaminergic system in thebrainstem as determined by a significant reduction in im-munoreactivity for TH in the NTS, DMV, area postrema,intermediate reticular nucleus, and VLM. These resultsare consistent with those of Storm and Fechter, 1985 (13)who reported a decrease in the concentration of noradren-aline in the pons/medulla in the newborn rat followingprenatal CO exposure. In the present study, the decreasein TH-IR in the VLM was associated with a significant

decrease in the number of TH-positive neurons in thisregion. Thus, the reduction in TH-IR is most likely large-ly due to this reduction in cell numbers although a de-crease in the content of the enzyme within neurons andtheir processes, a decrease in the size of neurons and theextent of their dendritic arbors, or a decrease in cate-cholaminergic input to the nuclei could also contribute.Although other noncardiorespiratory regions of the me-dulla show TH-IR (i.e. gracile, cuneate, and spinal tri-geminal nucleus) in both control and CO-exposed fetuses,the very sparse staining would not allow for accurate op-tical density measurements. At the level of qualitativeassessment there did not appear to be any changes inthese regions. It therefore appears that changes in TH-IRare seen in those regions of the medulla with a high cat-echolaminergic innervation. These are important regionsinvolved in cardiorespiratory control.

Since the nuclei mentioned above are involved in thecontrol of cardiac and respiratory activities, any alterationin the catecholamine content of their afferent and efferentconnections, or of the cell bodies themselves, could resultin abnormal functioning of these pathways during devel-opment and after birth. It is relevant that the major de-crease in TH-IR in the NTS was in the caudal extent ofthe nucleus, which receives chemosensory input (43–45).Consistent with this finding is our previous observationthat neonatal guinea pigs from CO-treated pregnanciesshow altered respiratory responses to asphyxia and CO2

(22).In relation to the cholinergic system, CO exposure af-

fected ChAT but not the m2 receptor expression in themedulla. Immunoreactivity for ChAT was significantlyincreased in the hypoglossal nucleus, and DMV, but notin the NTS or facial nucleus in CO-exposed fetuses com-pared with controls. The hypoglossal nucleus innervatesthe muscles of the tongue and is therefore important inthe process of swallowing. Although the hypoglossal nu-cleus is not generally considered to be a primary respi-ratory center, populations of hypoglossal neurons havebeen shown to discharge during inspiration, expiration,and transitional phases of respiration (46). An increase inChAT might directly or indirectly affect the control ofswallowing, in relation to breathing, in the neonate. It hasbeen suggested that the combination of impaired swal-lowing, deficient arousal mechanisms, and gastro-oesoph-ageal reflux can result in apnoea in the human infant (47–49). An interesting finding in the present study was thepresence of a population of m2-IR neurons located on theventral surface of the medulla. Studies have identified thepresence of a chemosensitive region here in neonatalguinea pigs and rabbits (50). In the fetal guinea pig, thisgroup of neurons appears to be located in a similar po-sition to the arcuate nucleus in the human (12) and therespiratory chemosensitive fields in the cat (51–53). Fur-thermore, muscarinic cholinergic receptor binding has

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TABLE 3Optical Density Measurements (M 6 SEM) for Substance P (SP) and Metenkephalin (ME)-immunoreactivity in the

Medulla of Control (n 5 8) Carbon Monoxide (CO)-exposed (n 5 8) Fetal Guinea Pigs

NucleiSP

Control CO-exposedME

Control CO-exposed

NTSXIIAPNTS cells (cells/mm2)MRF cells (cells/mm2)

0.20 6 0.010.09 6 0.010.21 6 0.038.6 6 2.5

—

0.19 6 0.020.09 6 0.010.22 6 0.038.8 6 1.2

—

0.08 6 0.010.04 6 0.010.07 6 0.01

—21.1 6 2.8

0.10 6 0.010.05 6 0.010.07 6 0.01

—19.7 6 2.7

Fig. 2. Western blot of tyrosine hydroxylase (TH) proteinpurified from the medulla of 62-day-old control and carbonmonoxide (CO)-exposed fetuses. Quantitative western analysisrevealed a 13% reduction in TH protein in the medulla of CO-exposed animals in comparison to controls. This result supportsquantitative immunohistochemical analysis.

←

Fig. 1. Bright-field photomicrographs demonstrating the distribution and intensity of tyrosine-hydroxylase-immunoreactivity(TH-IR) (panels A–D) and choline-acetyltransferase-immunoreactivity (ChAT-IR) (panels E and F) in the medulla of control(panels A, C, E) and carbon monoxide (CO)-exposed (panels B, D, F) fetal guinea pigs. Exposure to CO prenatally resulted ina significant decrease in the optical density of TH-IR in the nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus(DMV), ventrolateral medulla (VLM) (panel B), and area postrema (AP) (panel D) when compared with controls (panels A andC). There was also a decrease in the numerical density of TH-IR neurons in the region of the VLM and an apparent reductionin TH-IR in the intermediate reticular nucleus (IRt) of CO-exposed fetuses (panel B) when compared with controls (panel A).An increase in the optical density of ChAT-IR was measured in the hypoglossal nucleus (XII) and DMV of CO-exposed fetuses(panel F) when compared with controls (panel E). Calibration bar: (panels A and B) 5 34 mm; (panels C and D) 5 21 mm;(panels E and F) 5 22 mm. Abbreviations: 4V, fourth ventricle; cc, central canal; MRF, medullary reticular formation; ts, tractussolitarius; Xn, vagus nerve; XIIn, hypoglossal nerve.

been localized to the arcuate nucleus in the human infant(12).

In contrast to the effect of prenatal CO exposure onenzymes in the catecholaminergic and cholinergic sys-tems in the medulla, the levels of the neuropeptides SPand ME were not affected. This perturbation might affect

synthetic enzymes differentially from neuropeptides orthe effect might follow a different time course. As theanimals were exposed to CO over several weeks it wouldbe expected that if any affect were to have occurred itwould have been observed within this time frame.

Carbon monoxide may exert effects on the growingbrain by inducing hypoxia, although it is possible thatother mechanisms are involved as discussed below. Re-cent in vivo (54) and in vitro (55, 56) studies have shownthat short-term (,24 h) hypoxia mediates an increase inTH mRNA by increasing the rate of gene transcription.Following exposure to chronic hypoxia during pregnancyin rats, White and Lawson, 1997 (57) reported a decreasein TH mRNA and protein in the dorsal medulla at birth.After birth however, there was an increase in TH mRNAand protein, a decrease in PNMT protein in the dorsalmedulla, and an increase in TH mRNA in the ventralmedulla. Other studies investigating the effects of chronicpostnatal exposure to hypoxia have shown an increase inTH mRNA and/or protein in the caudal (58–61) and ros-tral NTS (59, 61), the DMV (61), the caudal dorsomedialmedulla (62), the VLM (61, 62) and the locus ceruleus(62). Therefore it appears that hypoxia has different ef-fects on the expression of tyrosine hydroxylase in thefetus, neonate, and adult.

There have been some studies on the effects of hypoxiaon the cholinergic system in the fetus, neonate, and adultand in general they reported a decrease in cholinergicmarkers across all ages. In a study of mouse fetal corticalcells, a decrease in ChAT activity was reported followingexposure to severe chronic hypoxia (63). A number of in

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vivo (64–67) and in vitro (68, 69) studies in the neonateand adult have shown that hypoxia suppresses the syn-thesis and presynaptic release of acetylcholine throughoutthe cerebral hemispheres. In these studies the only effectobserved in the brainstem was a reduction in ChAT-IRcells in the pedunculopontine nucleus following acute se-vere hypoxia (70). On the other hand, in studies using amodel that included hypoxia and ischemia, an increase inthe density of ChAT-IR in the neuropil (71), and neurons(72) in the striatum has been reported.

In addition to inducing hypoxia, it has been proposedthat CO has a direct cytotoxic effect (73, 74). Carbonmonoxide binds to many enzymes with a heme moiety,including mitochondrial cytochromes, thereby blockingenergy production and interfering with cellular respira-tion. Further, it is now known that CO is formed endog-enously in the nervous system by heme oxygenaseswhich oxidatively cleave the heme ring releasing CO andbiliverdin; CO has been implicated as a neuronal mes-senger activating soluble guanylate cyclase (75) and in-creasing the concentration of cGMP (76, 77). It is there-fore conceivable that the alterations in TH- and ChAT-IRseen in our study occur as a result of the exogenous COperturbing the neural pathways normally modulated byendogenously produced CO.

Thus, we have shown that chronic moderate prenatalCO exposure results in an increase in ChAT-IR in theDMV and hypoglossal nucleus and a decrease in TH-IRin the NTS, DMV, area postrema, intermediate reticularnucleus, and VLM in the medulla of the fetal guinea pig.The changes in TH-IR correlate well with studies in SIDSbrains where it has been reported that there is a decreasein the concentration of PNMT and DbH in the NTS,DMV, and the NAmb (20), and a reduction in TH-IR inthe vagal nuclei and the ventrolateral reticular formation(78). Aspects of the cholinergic system are affected inthe brainstem of SIDS infants, since it has been reportedthat there is a decrease in muscarinic receptor binding inthe arcuate nucleus (12). In this study, we found no dif-ference in the immunoreactivity of a specific muscarinicreceptor subtype (m2). The alteration observed by Kin-ney et al, 1995 (12) might therefore be attributed to dif-ferences in subtypes other than m2.

Alterations in the expression of neurotransmitters dur-ing fetal brain development will not only result in ab-normal neurotransmission, but might also influence neu-ronal differentiation and synaptic plasticity, since it isnow apparent that catecholamines (79–81) and acetyl-choline (80, 82–85) play a trophic role in brain devel-opment. Such effects could be of relevance to the devel-opment of circuitry in cardiovascular and respiratorynuclei in the neonate, and thus could have implicationsfor the relationship between cigarette smoking duringpregnancy and abnormal brain development.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. Michelle Loeliger for her excellenttechnical assistance and Mrs. Kerryn Westcott for her involvement inthe generation and care of the animals used in this study. We wouldalso like to acknowledge Drs. Brian Key, James St. John, Richard An-derson, and Heidi Clarris for their help with the western blot analysis.

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Received October 4, 1999Accepted November 30, 1999