Is There a Zone of Vascular Vulnerability in the Fetal Brain Stem?

Neurotoxicology and Teratology, Vol. 19, No. 4, pp. 265–275, 1997

Copyright

©

1997 Elsevier Science Inc.Printed in the USA. All rights reserved

0892-0362/97 $17.00

1

.00

PII S0892-0362(97)00020-2

265

Is There a Zone of Vascular Vulnerabilityin the Fetal Brain Stem?

SHARON LEONG AND KEN W. S. ASHWELL

School of Anatomy, The University of NSW, 2052, NSW, Australia

Received 14 October 1996; Accepted 23 February 1997

LEONG, S. AND K. W. S. ASHWELL.

Is there a zone of vascular vulnerability in the fetal brain stem?

NEUROTOXICOLTERATOL

19

(4) 265–275, 1997.—The pattern of malformations in congenital anomalies such as Möbius syndrome and fol-lowing prenatal cocaine exposure suggests that there is a zone of vascular vulnerability or ischemic sensitivity in the parame-dian region of the developing brain stem. In the present study, postmortem examination of the brain of an infant with Möbiussyndrome revealed mineralized foci concentrated in paramedian wedge-shaped areas of the pontine and medullary tegmen-tum. We also examined the development of brain stem vasculature in the rat at the light and ultrastructural level to determinewhether anatomical features of the paramedian brain stem region could contribute to elevated incidence of vascular accidentsin that zone. Several observations of relevance to the question of vascular vulnerability of the midline were made. Firstly, andas previously noted by other authors, the brain stem midline remains avascular for protracted periods during fetal life. Wepropose that the inability of vessels in the paramedian region to anastomose across the avascular midline gives rise to para-median watershed zones that could be vulnerable to ischaemia in the event of hypoperfusion due to teratogenic action. Sec-ondly, we studied the development of cytochrome oxidase activity in the fetal brain stem and noted high oxidative metabolicactivity of the somatic efferent nuclei in the paramedian region, which could render their constituent neurons particularly sus-ceptible to hypoxia. Thirdly, our ultrastructural examination revealed large amounts of extracellular space surrounding para-median pontine vessels in comparison to laterally placed vessels, although there was no significant difference between thevessels of the two regions in tight junction length and endothelial thickness. We propose that the greater proportion of unoc-cupied extracellular space surrounding medial vessels may contribute to poorer support of these vessels in ischemic/reperfu-

sion episodes. This poor support could in turn give rise to an increased risk of hemorrhage.

©

1997 Elsevier Science Inc.

Möbius syndrome Maternal cocaine abuse Uterine trauma Hyperthermia

BRAIN hemorrhage and infarction have been noted in sev-eral congenital malformations and even among apparentlynormal fetuses (3). For example, calcification of, and gliosisaround, the dorsal respiratory group of nuclei (located in theupper medial third of the dorsal pons) is common in infantswho die from respiratory arrest (11,26,30), and some investi-gators have noted brain stem vascular abnormalities and glio-sis associated with the Sudden Infant Death Syndrome (29).Vascular abnormalities in somatic tissue have also been re-ported in some congenital anomalies. The Subclavian ArterySupply Disruption Sequence (4,26), which involves disruptionto the embryonic blood supply at specific locations in the sub-clavian arteries and their branches, is thought to underlieKlippel-Feil syndrome, Poland syndrome, Möbius syndromeand Sprengel deformity. These syndromes present with sev-eral abnormalities, including defects of the cervical vertebrae,pectoralis musculature, and shoulder girdle, as well as palsies

of the facial and other cranial nerves. The occurrence of thefeatures of these syndromes together in some patients has ledinvestigators to consider that these syndromes are related(4,26), the common element being the dependence of all the af-fected structures upon the subclavian artery and its branches.

The Möbius syndrome is characterised clinically by a group ofcranial nerve palsies (particularly facial, abducens, hypoglossal,although oculomotor may also be involved) (4,6,8,11,17,26,30).Histopathological examination of the brain stem often revealsunilateral or bilateral hypoplasia or necrosis of the abducens andfacial nuclei, accompanied by gliosis, calcification, and enlargedperivascular space (8,30). Experimentally, both the brain stemfeatures and the limb abnormalities have been mimicked byclamping the uterine vessels in animal models (10,18,19,31,32).Furthermore, animal models of vasoactive substance abusehave also induced vascular injury in the brain (16,33,34), withinthe midline regions of the midbrain tegmentum as well as the

Requests for reprints should be addressed to Dr. Ken W. S. Ashwell, School of Anatomy, The University of NSW, NSW, 2052, Australia.

266 LEONG AND ASHWELL

striatal anlage. Although the rostrocaudal position of hemor-rhage is different in the two models (uterine clamping vs. drugexposure), the hemorrhages typically occur near the midlinein both.

The question arises then: why does alteration of bloodpressure give rise to vascular injury in particular parts of thebrain stem? In our study we have firstly examined the distri-bution of injury in the brain stem of an infant with Möbiussyndrome, to confirm that the pattern of damage is indeedconcentrated in the paramedian region, as expected from theclinical features of this syndrome.

Subsequently, we have tested several hypotheses concern-ing the apparent vulnerability of the paramedian zone of thebrain stem tegmentum. This latter part of the investigationhas been undertaken in animals due to the lack of suitablepostmortem human material of the relevant age. Rodentswere chosen for this analysis because they have been used inseveral models for both Möbius syndrome and maternal co-caine abuse (19,31,32).

The hypotheses we considered were: i) that the midlineand paramedian zone of the developing brain stem tegmen-tum is poorly vascularized relative to the more lateral regionsof the same structure, ii) that the midline and paramedianzone of the brain stem contains a higher proportion of imma-ture vessels (as assessed by growing tip density), which couldbe prone to hemorrhage in the event of ischemic/reperfusionepisodes, iii) that the ultrastructural features of vessels in theparamedian zone (specifically tight junction morphology andperivascular support) may be immature relative to those inmore lateral regions, and iv) that the metabolic demand of themidline paramedian zone is high, with the potential to exceedthe available supply during ischemic episodes. This last hy-pothesis was suggested by the finding that cranial nerve motornuclei are sensitive to low pressure and reperfusion in adults(12). Such sensitivity would not contribute to the apparentlyhigher rate of hemorrhage and leakage, but might be an im-portant factor in the degree of damage sustained during is-chemic episodes.

METHOD

Brain stem sections of an infant with Möbius syndromewere kindly made available by Professor Robert Osborne ofThe Institute of Clinical Pathology and Medical Research atWestmead Hospital. The infant was a female, who died at theage of 7 months from aspiration and respiratory arrest. Exter-nal features of this infant included syndactyly of the left handand foot, absent right hand, and shortened digit 1 of the rightfoot. The brain stem had been immersion fixed in formalin, di-vided into coronal slabs at the levels of cranial nerves IIIthrough to XII, embedded in paraffin, and sectioned. These sec-tions were analysed using a computer-linked microscope run-ning the Magellan 5.3 program. The distributions of mineralizedfoci, as well as major nuclei and tracts where discernible, weremapped out throughout the rostrocaudal extent of the brainstem, with reference to an atlas of the human brain stem (23).

Animal experiments were undertaken using the Wistarstrain of rats. Females were mated overnight and the day offinding a sperm-positive vaginal smear was designated E0(embryonic day 0) of gestation. Dams were overdosed by IPinjection of pentobarbitone sodium (60 mg/kg) at E11 to E18,and the fetuses distributed into groups for lectin and cyto-chrome oxidase histochemistry or electronmicroscopy.

We have chosen lectin histochemistry with the

Griffoniasimplicifolia

B

4

isolection to reveal vasculature because it does

not require invasive procedures such as vascular perfusion.With prolonged incubation the lectin penetrates thick sectionswell, allowing the use of relatively thick sections, which revealangioarchitecture clearly. When combined with peroxidase la-bel a high contrast, permanent preparation is obtained. Al-though the lectin also labels microglia, these are easily distin-guished from vessels, because the former are small androunded or with short processes whereas the latter show lu-mina and intravascular erythrocytes, and extend for long dis-tances (1). For lectin histochemistry, three to four fetuses ateach age were fixed by immersion in 4% paraformaldehyde/1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4

°

Covernight. Tissue was cryoprotected overnight at 4

°

C in 30%sucrose in 0.1 M phosphate buffer (pH 7.4). Photomicro-graphs of the lateral brain stem were taken to aid with sectionalignment of reconstructions. The brain stems were frozen-sectioned coronally at a thickness of 15

m

m (E11) or 120

m

m(E12 to E18). Sections were immersed overnight at 4

°

C in asolution of horseradish-peroxidase-conjugated lectin (

Grif-fonia simplicifolia

, B

4

isolectin, 0.5 mg/100 ml in Tris-bufferedsaline with 1% Triton X-100) obtained from Sigma ChemicalCompany. This product has given highly specific and repro-ducible results for the decade over which our group has usedit (1). The appropriate control consists of incubating the lectinwith the sugar melibiose, which blocks labelling of vessels andmicroglia (1). Sections were rinsed and reacted with thenickel-enhanced DAB technique, air dried, and coverslipped.Linear shrinkage of the sections was minimal (less than 5%)and uniform. Three-dimensional reconstructions of the brainstems were made using the Magellan 5.3 program, indicatingthe positions of vascular branch points and growing vascularsprouts. Identification of nuclei was made by reference to anatlas of the developing rat nervous system (22).

Quantitative analysis of vasculature was made by countinggrowing tips and branch points in volumes 200

m

m on a sideand 120

m

m thick (volume of 4.8

3

10

6

m

m

3

) from medial andlateral parts of the pontomedullary junction neuroepithelium.These zones for analysis were arranged with one side alongthe ventricular surface. Three samples at the medial and lat-eral zones were analysed per animal using three animals perage group (E13, E14, E15, and E16). Statistical comparisonswere made of the densities of branch points and growing tipsin medial and lateral tegmentum with the aid of the Mann–Whitney

U

-test. This ranking test was chosen because a nor-mal distribution of the variables could not be guaranteed withthe small samples available.

For electron microscopy, three fetuses at each age wererapidly removed and fixed by immersion at 4

°

C in 2% para-formaldehyde/2% glutaraldehyde in 0.1 M cacodylate buffer.With the aid of a dissecting microscope, slabs were removedfrom the pontomedullary junction and the blocks postfixedovernight at 4

°

C in the same fixative. Blocks were osmicatedfor 1.5 h at 4

°

C, block-stained in 2% uranyl acetate, dehy-drated, and embedded in Durcupan (Fluka). Semithin sec-tions (0.5–0.9

m

m thickness) and ultrathin sections (60–80 nmthickness) were cut with the aid of Reichert-Jung Ultracut Eand stained with toluidine blue or 1% lead citrate, respec-tively. Ultrathin sections were examined with the aid of an Hi-tachi H-7000 transmission electron microscope, and electron-micrographs taken at magnifications of 5,000

3

for assessingextracellular space and general vessel morphology or 20,000

3

for quantitative assessment of tight junction morphology. Quan-titative analysis of extracellular space surrounding vessels wasmade by laying a grid of points at 0.25-

m

m intervals across thelow-power micrographs (final magnification of 15,000

3

) and

FETAL BRAIN STEM VASCULATURE 267

counting the number of points lying in extracellular space sur-rounding vessels in the medial and lateral tegmentum. Be-tween 9 and 22 sample zones (2.5 by 2.5

m

m in size) arrangedaround these vessels were analysed for each age group and re-gion from three animals at each age. Tight junction morphol-ogy was assessed on high-power electronmicrographs. Thelength of the entire junctional region and the length of thejunctional region where tight membrane apposition was ap-parent and were measured with the aid of the Magellan 5.3program. Statistical comparisons were made at each age be-tween vessels from medial and lateral pontomedullary junc-tion of the tegmentum with the aid of the nonparametricMann–Whitney

U

-test.We were interested in comparing oxidative metabolic ac-

tivity in the medial and lateral parts of the developing ratbrain stem. For this purpose, cytochrome oxidase histochem-istry was performed on three to four fetuses at each of theages E11, E12, E15, E16, and E19. Fresh tissue was immersedin Tissue Tek (Miles) filled small aluminium boats, whichwere rapidly frozen in liquid nitrogen. The brain stems weresectioned coronally with the aid of a cryostat at a thickness of15

m

m. Sections were collected onto 5% gelatin-coated slides,which were briefly air dried before incubation to demonstratecytochrome oxidase activity according to the Wong-Riley tech-nique (35). Incubation was carried out at 38

°

C for 20 h toachieve maximal contrast between neural tissue and back-ground. Sections from all ages were incubated together in thesame batch of medium to ensure uniform reaction parameters.Control sections were incubated without the substrate cyto-chrome C; activity present in the absence of the substrate is

considered to be due to endogenous peroxidase activity (35).Slides were subsequently coverslipped and photographed.

RESULTS

Distribution of Mineralized Foci in the Brain Stem of the Möbius Infant

Evidence of necrosis in the brain stem of the Möbius syn-drome infant, as revealed by the presence of mineralized fociand gliosis, was distributed from the caudal pontine tegmen-tum through to the caudal medullary tegmentum. These fociwere concentrated in paramedian strips throughout this ros-trocaudal extent (see Figs. 1 and 2). Within the pons, mineral-ized deposits were present bilaterally within the presumptivedorsal paragigantocellular nuclei and extending as far later-ally as the intermediate reticular zone. The mineral depositswere also found in the region of the abducens nucleus and in-ternal genu of the facial nerve. Deposits were scattered through

FIG. 1. Photomicrograph of mineralized foci (double arrow) withinthe rostral medulla of an infant with Möbius syndrome (see diagramin Fig. 2C). Hematoxylin and eosin stain. 4V, fourth ventricle.Magnification: 153.

FIG. 2. Drawings of a series of sections (A, rostral pons; B,pontomedullary junction; C, rostral medulla; D, caudal medulla)through the brainstem of the infant with Möbius syndrome showingthe paramedian distribution of mineralized foci (black filling). c, nucleuscuneatus; g, nucleus gracilis; io, inferior olivary nuclei; pn, pontinenuclei; v, nucleus of trigeminal spinal tract; x, dorsal motor nucleus ofthe vagus; xii, hypoglossal nucleus. Magnification: 3.53.


the region of the presumptive medial lemniscus (midline re-gion of Fig. 2).

At the pontomedullary junction, mineralized foci wereonce again found in the gigantocellular nuclear complex (notshown) and medial lemnisci. Further caudally, in the rostralmedulla, there was evidence of involvement of the raphe nu-clear complex (nucleus raphe obscurus and paramedian raphe),and hypoglosal nerve, with degeneration and mononuclear in-flammatory cell infiltration of the hypoglossal nucleus.

Lectin Histochemistry of Developing Brain Stem Vasculature

Initial vascular invasion of the brain stem was apparent atE11 (not shown), but these early vessels had not entirely tra-versed the substance of the brain stem until E12. At that age,vessels invading the brain stem were concentrated along theventrolateral aspect of the hindbrain. Invading vessels pene-trated these parts of the brain stem in a radial direction for35 to 50

m

m before forming a one-tiered plexus parallel to theventricular surface (Fig. 3A, B). Some regions of the brainstem were avascular at this stage. These included the dorsalneural tube adjacent to the roof of the developing fourth ven-tricle, and the ventral midline, which corresponds to the floorplate of the neural tube (Fig. 3A, B). Rostral mesencephalonwas relatively poorly vascularized compared to the ventrolat-eral hindbrain. Only a few canalised vessels were evident inthis region; blood supply to the ventral and dorsal mesenceph-alic midline was from plexuses derived from more laterallyarising stem vessels. These stem vessels were up to 200

m

mfrom the midline regions.

By E13 the perineural vascular plexus formed a densesheath around the entire brain stem. Several areas remainedavascular at this age. These include the dorsal mesencephalonand ventral isthmus. At E14, these avascular zones remainedin the dorsal mesencephalon, where a 50-

m

m-wide avascularregion was apparent and along the ventral midline of the ponsand medulla (a region 85–100

m

m wide) (Fig. 3C). In one E14fetus (Fig, 4A, B, C), evidence of microhemorrhage was foundin the midline of the isthmus, but not laterally.

By E15, the midline of the pontine and medullary tegmen-tum remained avascular, but the ventral midline of the mesen-cephalon, containing the developing dorsal raphe nuclei, be-gan to receive direct vascular supply. At this age feeder orstem vessels were first seen crossing the midline of the dorsalmesencephalon. The pattern of feeder or stem vessels to thebrain stem was similar at E16 to E18, compared to E15. Theventral midline of the pontine and medullary tegmentum re-mained avascular until the last age examined (E18).

In several E16 fetuses, we noticed evidence of spontaneoushemorrhage (Fig. 4D, E). These were noted in four animalsout of four examined, and were found in the ventral midlineof the developing mesencephalon. No microhemorrhage wasseen laterally within the brain stem tegmentum.

Quantitative Analysis of Branch Point and Growing Tip Density During Brain Stem Development

Quantitative analysis of branch point and growing tip den-sity was confined to the ages E13 to E16, because the vascularplexus is still incomplete at E12. The results are summarisedin Table 1.

Branch point density, both medially and laterally, tendedto rise with increasing gestational age. At E14 to E16, thebranch point density of the medial pontomedullary tegmentalneuroepithelium was slightly higher than that for the lateralneuroepithelium. Growing tip density was highest at E14 and

FIG. 3. Lectin histochemical staining of blood vessels in thedeveloping rodent brainstem. (A) Vasculature of the mesencephalonat E12, showing avascularity of the ventral (open arrowhead) andpoor vascularity of the dorsal (curved arrow) midline of the futuremidbrain. (B) Section through the brainstem of an E12 fetus showingan avascular region (open arrowhead) in the midline of therhombencephalon at the level of the otic vesicle. (C) The avascularmidline in the pons (above) and medulla (below) at E14. 4V, fourthventricle. Magnifications: (A, B) 453; (C) 203.


E15, but at none of the ages examined was there any signifi-cant difference between the medial and lateral tegmental neu-roepithelium in growing tip density.

Electron Microscopy of Developing Brain Stem Vasculature

At E11 (not shown), most vessels associated with the brainwere confined to the perineural vascular plexus. The only in-traparenchymal vessels present showed thin walls (0.1–0.7

m

mthick) with abundant pinocytotic vesicles within the endothe-

lium on both the luminal and abluminal sides. In contrast toreports of early intracerebral capillaries (25,26,36), electron-dense tight junctions were well developed and fenestrationswere absent from these early brain stem capillaries. Verylarge extracellular spaces were apparent surrounding the ves-sels, and glial elements were absent at this age.

By E12, vascular invasion of the neural tissue was well ad-vanced (Fig. 5). The ultrastructural features of these earlybrain stem vessels included well-developed endothelial walls,with the interendothelial junctions showing several discontin-

FIG. 4. Spontaneous hemorrhage (arrows) in the ventral paramedian zone of the pontine isthmus ofan E14 fetus (A, B, and C low and progressively higher powers) and the ventral median midbrain ofan E16 fetus (D and E, low and high power). Arrows indicate microhemorrhages in (A), (B), and(C), and arrowhead indicates hemorrhage in (D); (E) shows a high-power view of microhemorrhagearrowed in (D). Magnifications: (A, D) 153; (B, E) 453; (C) 1503.


uous tight junctions along their length. Marginal folds wereapparent at the luminal end of the interendothelial junctions.Micropinocytotic vesicles (of 0.2-

m

m diameter or less) weredistributed throughout the endothelial cytoplasm. A few glialprocesses had developed between the neuroepithelial cells,but these processes had not extended to the capillary walls.As at E11, there was a large amount of extracellular space,both medially (26.2%) and laterally (28.3%) within the pon-tomedullary tegmentum.

From E13, pericytic processes were first seen surroundingendothelial cells. Also associated with endothelial cells ofboth medially and laterally located vessels were glioblasticprocesses, which could be identified by an electronlucent cy-toplasm and large vesicles of 0.2

m

m diameter or greater.Tight junction morphology was similar to E12; most interen-dothelial junctions were characterised by tight junctions ex-tending for up to 75% of the length of the apposing mem-branes. A noticeable feature of brain stem ultrastructure atthis age was the difference between the proportion of emptyextracellular space in the medial and lateral pontomedullarytegmentum. Medial tegmental tissue elements were surroundedby empty extracellular space occupying on average 18.9% ofthe electronmicrograph area, whereas in the lateral tegmen-tum of the same animals only 5.8% was empty extracellularspace (Table 2). At higher magnifications, sparsely distributedelectronlucent flocculent material between the abluminal en-dothelial cell membrane and the pericytic processes was seenfor the first time (not shown). This resembled the earliest evi-dence of a developing basal lamina as described in the fetalcerebral cortex by other authors (5,15).

At the subsequent ages examined (E14, E15, and E16),glioblastic processes were seen with increasing frequency con-tacting parenchymal capillaries, and the basal lamina in-creased in density. At both E14 and E15, as noted previouslyat E13, vessels located in the medial tegmentum were sur-rounded by increased extracellular space (36.3% at E14;28.6% at E15) compared to laterally situated vessels (13.1%at E14; 5.0% at E15). Figure 6 shows examples of laterallyand medially situated vessels in an E15 fetus. It was also notedthat lateral tegmental vessels were better supported by glio-blast processes compared to medial tegmental vessels. AtE16, the aforementioned differences between the perivascularspace surrounding medial and lateral tegmental vessels werestill apparent (40.5% medially compared to 15.3% laterally).

Parenchymal vessel wall thickness was relatively high dur-ing early invasion of the brain stem (mean

6

SD, E11—0.29

6

0.15

m

m; E12—0.23

6

0.09

m

m; E13—0.35

6

0.15

m

m; E14—0.22

6

0.09

m

m; E15—0.22

6

0.09

m

m), before dropping atE16 (0.16

6

0.07

m

m). We were unable to identify any signifi-cant differences between medial and lateral pontomedullarytegmental vessels in terms of vessel thickness.

Quantitative Analysis of Tight Junction Morphology During Brain Stem Vasculature Development

We were interested in determining whether there were anydifferences in tight junction morphology between medial and

TABLE 1

DENSITY OF BRANCH POINTS AND GROWING TIPS INMEDIAL VS. LATERAL PONTINE TEGMENTUM

Age

Branch Point Density(mean

6

SD; Nbp/10

6

m

m

3

)Growing Tip Density

(mean

6

SD; Ngt/10

6

m

m

3

)

Medial Lateral Medial Lateral

E13 2.66

6

0.80

NS

2.11

6

0.29 0.66

6

0.08

NS

0.71

6

0.27E14 3.33

6

0.52* 2.45

6

0.77 1.01

6

0.37

NS

1.04

6

0.08E15 4.25

6

0.89† 2.74

6

0.52 0.89

6

0.35

NS

0.67

6

0.35E16 3.92

6

0.56† 2.31

6

0.66 0.44

6

0.27

NS

0.23

6

0.16

NS

Not significant,

p

.

0.10, Mann–Whitney test.*Significant difference,

p

,

0.005, Mann–Whitney test.†Significant difference,

p

,

0.001, Mann–Whitney test.FIG. 5. Electronmicrograph of a vessel in the fetal brainstem. AtE12, the vessel lumen is irregular, tight junction formation in theintraparenchymal vessels is already advanced (double arrow).Magnification: 10,0003.

TABLE 2

EXTRACELLULAR SPACE AS ASSESSED BY ELECTRON MICROSCOPY

Age

Medial Lateral

No. Samples %x

6

SD % Range No. Samples %x

6

SD % Range

E12 9 26.2

6

14.5

NS

8–58 18 28.3

6

16.8 0–68E13 16 18.9

6

15.9* 0–54 18 5.8

6

6.7 0–22E14 19 36.3

6

22.3† 8–100 23 13.1

6

16.3 0–68E15 17 28.6

6

15.1† 13–72 22 5.0

6

3.2 0–14E16 19 40.5

6

16.9† 17–68 13 15.3

6

19.8 0–72

NS

Not significant,

p

.

0.10, Mann–Whitney test.*Significant difference,

p

,

0.005, Mann–Whitney test.†Significant difference,

p

,

0.001, Mann–Whitney test.


lateral tegmental vessels at the pontomedullary junction. Fig-ure 7 shows tight junction morphology and Table 3 shows thequantitative results from this analysis.

There was considerable variation in length of the interen-dothelial junction, length of tight junction, and proportion ofthe interendothelial junction that was tight within a given age,but there was a general trend towards an increase in the pro-portion of the interendothelial junction that was tight withincreasing age. Thus, among the lateral tegmental vessels, thisproportion increased from 34.3% at E12 to 52.4% at E16. Asimilar trend was apparent in the medial tegmentum, risingfrom 36.0% at E12 to 48.5% at E15 and 44.2% at E16. Therewere no significant differences between medial and lateraltegmental vessels in any of the three parameters examined(

p

.

0.05).

Cytochrome Oxidase Histochemistry of Fetal Brain Stem

Cytochrome oxidase activity at all ages was localised infine puncta within the cytoplasm of the embryonic and fetalbrain stem tissue. These presumably represent labelling of theouter layer of the inner mitochondrial membrane (35). Con-trol sections (i.e., those sections incubated without cyto-chrome C) did not show these puncta, and background label-ling was diffuse, indicating no specific labelling.

At E11, there were no specific areas of higher oxidative ac-tivity within the fetal brain stem, although the ventricular sur-face of the mesencephalon showed higher activity than theouter ventricular germinal zone at that level. By E12, bands ofhigher oxidative activity were apparent along the ventricularand pial surfaces of the brain stem neuroepithelium, in partic-ular along the paramedian region where the developing ocu-

FIG. 6. At E15, a vessel located in the lateral pontine tegmentum (A) surrounded by abundant glial, pericyticand axonal processes. By contrast, the vessel from the medial pontine tegmentum shown in (B) is surrounded bylarge regions of empty extracellular space, outside the pericytic envestment (P). Magnification: 12,0003.


lomotor and trochlear nuclei are to develop (Fig. 8A, B). Oxi-dative activity in the developing myelencephalon at this agewas also concentrated along the paramedian region (Fig. 8C), inthe region of the developing somatic efferent nuclear column.

By E15, there was an overall increase in oxidative activitycompared to previous ages, with regions of particularly highdensity in the oculomotor and Edinger Westphal nuclei (notshown), hypoglossal nuclei (XII in Fig. 8D), and the dorsalmotor nuclei of the vagus. By this age, the ventral midline ofboth the mesencephalon and myelencephalon (incorporatingthe dorsal raphe, median raphe, raphe magnus, and raphe ob-scurus nuclei) was distinctly less active compared to more lat-eral tegmental regions (not shown). A similar pattern of stain-ing was observed at E16, except that staining intensity in the

ventricular neuroepithelium began to decline compared tosurrounding tegmental tissue.

The relatively low density of staining in the ventral midlinewas still apparent at E19 (not shown). By this age the most in-tensely staining regions were the oculomotor nucleus in themidbrain; the facial, abducens, and ventral nucleus of the lat-eral lemniscus in the pons; and the hypoglossal nucleus anddorsal motor nucleus of the vagus in the medulla.

DISCUSSION

Is there a Medial Zone of Vascular Vulnerability in the Fetal Brain Stem?

As outlined in the Introduction, several lines of evidencesuggest that the midline and paramedian zones of the devel-oping brain stem are particularly sensitive to vascular acci-dents. This evidence includes the pattern of affected cranialnerve nuclei in Möbius syndrome and the pattern of hemor-rhage in experimentally induced prenatal cocaine exposure.Some of the findings of the present study are in support of thisproposition. These include the striking paramedian distribu-tion of mineralized foci in the brain stem of the infant withMöbius syndrome, and the presence of spontaneous hemor-rhage in the midline of the brain stem observed in some of ourrodent fetuses.

Is the Paramedian Brain Stem Tegmentum Relatively Poorly Vascularized During Development?

It was originally hypothesised that there are significant dif-ferences between the vasculature of the medial and lateralbrain stem tegmentum, which might account for increasedsusceptibility of the midline and paramedian region to vascu-lar accidents. With regard to this, it was stated in our first hy-pothesis that there might be a reduced density of vasculaturein the midline and paramedian region of the brain stem duringdevelopment. Such hypovascularity could contribute to inade-quate perfusion or hypoxia of the paramedian region in theevent of circulatory disturbance or general hypoxia. Thepresent results indicate that there are indeed several regionsof the developing brain stem that are relatively poorly vascu-larized at some early stages of development. These includethe dorsal and ventral midline of the mesencephalon, and themedian raphe region of the pontomedullary junction. Theventral mesencephalon receives a vascular supply at E13,within 24 h of the lateral brain stem, whereas the dorsal mes-encephalon remains avascular until E15, a further 48 h. Themidline raphe of the pontomedullary junction remains avas-cular until at least E18, although occasional anastomotic ves-sels were seen crossing the midline after this age.

Are There More Immature Vessels in the Paramedian Region?

We suggested in our second hypothesis that there might bea preponderance of immature vessels in the midline region ofthe brain stem that might contribute to increased likelihood ofhemorrhage in this area. We had originally posited that such aconcentration of immature vessels could make this area moreprone to hemorrhage in the event of fluctuating perfusionpressure, which might accompany uterine trauma and manip-ulation. Goldberg et al. (14) have proposed that hypotensionfollowed by blood volume reexpansion may cause intraven-tricular hemorrhage in preterm infants. Indeed, clinically rele-vant animal models of the latter and of acute hypertension inearly postnatal animals have readily produced hemorrhage inthe ventricular germinal zone of the forebrain (13,20). In the

FIG. 7. Tight junction (curved arrow) morphology in the developingbrain stem at E12 (A) and E14 (B, C). Note the density of apposedmembranes. Magnification: 44,0003.


present study these immature vessels could be identified in lec-tin-stained material by their fine filopodial extensions, but ourquantitative analysis showed that, contrary to expectations,there was no particular concentration of immature vessels inthe paramedian region. Thus, we must reject our original hypoth-esis that the concentration of immature vessels in this regioncould contribute to the vulnerability of the medial tegmentum.

The Ultrastructural Development of Brain Stem Vasculature: Are There Differences Between Medial and Lateral Tegmentum?

In the present study it was found that brain stem capillariesare quite mature from the earliest stages of invasion of theneuraxis. Tight junctions were apparent from E11 and fenes-trations were absent from the earliest vessels. Previous re-ports on the development of intracerebral capillaries havenoted endothelial fenestrations as late as E17 in the rat cere-bral cortex (36). Stewart and Hayakawa (27) and Risau andcoworkers (24,25) give slightly earlier ages for the maturationof the blood–brain barrier in rat cerebral cortex (i.e., E13 andE15, respectively). Clearly, brain stem vessels are consider-ably advanced compared to cerebral vessels with respect tothese ultrastructural features of blood–brain barrier matura-tion. Stewart and Hayakawa (27) also report progressivetightening of tight junctions during vessel development in therat cerebral cortex.

Development of the basal lamina and investment of capil-laries by pericytes were also advanced in the brain stem com-pared to reports of cerebral vessel development. Both peri-cytes and the basal lamina appeared at E13 in vessels of thepontomedullary junction, but in the cerebral cortex pericytesdo not appear until E15, and the basal lamina at E17 (2,21).

At the outset of this investigation we proposed that theremight be regional differences in capillary ultrastructure be-tween medial and lateral pontomedullary tegmentum, whichcould contribute to an increased likelihood of hemorrhage orhypoperfusion ischaemia in the midline and paramedian re-gion of the fetal brain stem. Several ultrastructural features ofdeveloping vessels were examined with respect to this notion.These included: endothelial cell thickness, interendothelialjunction morphology (total length of the interendothelialjunction, actual length of the junction which was tight, and theproportion of the length of the interendothelial junctionwhich was tight), and the degree of support for the growingvessels provided by surrounding tissue elements (e.g., glio-blasts, pericytes, and the basal lamina).

The present findings show that there are no significant dif-ferences between the medial and lateral parts of the pon-

tomedullary junction in endothelial cell thickness, interendo-thelial junction morphology, or the time course of developmentof the basal lamina. There was, however, a noticeable differ-ence between the medially and laterally placed vessels in theproportion of the extravascular space occupied by potentiallysupportive elements. Thus, at E13, E14, E15, and E16 therewere fewer glioblastic processes surrounding the medially lo-cated vessels than seen around the vessels located laterally. Itis possible that relatively poor support of vessels during earlyfetal life could contribute to an increased risk of these vesselsundergoing rupture during hypoxic episodes and hypoperfu-sion followed by reperfusion as proposed for the forebrain (14).

The High Oxidative Metabolic Demands of Nuclei in the Paramedian Tegmentum

Clearly, consideration should also be given to the meta-bolic demands of poorly vascularized regions of the develop-ing brain stem. If metabolic demands are high in those re-gions, then the likelihood of ischemic necrosis during periodsof poor fetal perfusion is increased. The findings of thepresent study, with respect to cytochrome oxidase activity,throw some light onto this question. Cytochrome oxidase ac-tivity was relatively high in the ventral paramedian region ofthe mesencephalon at E12. This coincided with relativelypoor vascularity of this region at this age (see above). Such afinding could suggest that this region is prone to ischemic in-jury. The findings of Webster et al. (34), who used maternalcocaine administration and uterine vessel clamping, suggestthat this region does undergo ischemic necrosis following ar-terial vasoconstriction, but their study was carried out withslightly older fetuses (E16).

Watershed Zones in the Developing Brain Stem

Perhaps of as much significance as the avascular regions ofthe developing brain stem are the watershed zones. Classi-cally, vascular supply to the adult mammalian brain stem isderived from three groups of stem vessels. These are ventraland lateral groups in the pons and medulla above the level ofthe gracile and cuneate nuclei, and a further posterior groupin addition to those two in the midbrain (7). Other authorshave categorised the groups into ventral, ventrolateral, andlateral penetrating stem vessels (9,28). The developing brainstem is supplied initially by stem vessels penetrating the ven-trolateral aspect of the medulla, pons, and midbrain. Inslightly older fetuses (E15 in the present study), stem vesselspenetrate the lateral aspects of the medulla, pons and mid-brain and the ventral surface of the midbrain. As a conse-

TABLE 3MORPHOLOGY OF BRAINSTEM INTERENDOTHELIAL (IE) JUNCTIONAL COMPLEXES

Age

Medial Lateral

Mean Length ofIE Junction (A)

(mm 6 SD)

Mean Length ofTight Junction (B)

(mm 6 SD)B/A(%)

Range of B/A(%)

Mean Length ofIE Junction (A)

(mm 6 SD)

Mean Length ofTight Junction (B)

(mm 6 SD)B/A(%)

Range of B/A(%)

E12 2.48 6 0.84NS 0.94 6 0.49NS 36.0 22.0–46.0 2.12 6 1.19 0.63 6 0.40 34.3 12.4–76.8E13 1.78 6 0.93NS 0.77 6 0.52NS 42.4 24.6–58.7 1.76 6 0.75 0.66 6 0.33 38.8 21.2–66.6E14 1.63 6 0.73NS 0.81 6 0.58NS 47.2 24.9–72.3 2.29 6 1.04 0.74 6 0.38 35.5 10.6–71.7E15 1.70 6 0.40NS 0.86 6 0.44NS 48.5 26.7–80.1 2.36 6 1.15 0.90 6 0.38 45.9 9.3–66.2E16 1.73 6 0.80NS 0.80 6 0.52NS 44.2 22.3–70.1 1.49 6 0.41 0.76 6 0.30 52.4 23.5–77.2

NSDenotes no significant difference between medial and lateral vessels in this parameter.


quence of this sequence and the absence of vascular anasto-moses across the midline, the paramedian zones of the fetalbrain stem are dependent for most of the late prenatal lifeupon anastomotic branches passing medially from the ventro-lateral stem vessels. In other words, the paramedian regionscan be considered to receive end-vascular supply. Becausesome of these end-vessels are not yet patent, this region may bevulnerable to ischaemia during hypoperfusion, and, for this rea-son, the paramedian zones may be defined as watershed zones.This arrangement is summarised in Fig. 9. These watershedzones extend dorsally to include the somatic efferent nuclearcolumn (e.g., abducens and hypoglossal nuclei). In the adult,both the midline and periventricular region of the somatic ef-ferent nuclei have been documented as receiving end-vascularsupply (7). We would like to propose that this watershed zoneis particularly vulnerable to hypoperfusion.

Concluding Remarks

In conclusion, the sequence of vascular developmentalevents in the fetal brain stem is similar to that seen in otherparts of the developing neuraxis [e.g., cerebral cortex (2, 5, 36)],but occurs several days earlier. Several features of early brainstem vascular development may contribute to an increased riskof ischemic necrosis and/or hemorrhage in the midline of thefetal brain stem. These include: i) prolonged avascularity of themidline regions of the brain stem, with the paramedian zonealso becoming at risk due to the watershed nature of its bloodsupply, ii) increased extracellular space surrounding medialtegmental vessels compared to their more lateral counterparts,with consequently poorer glioblastic support and a possibly in-creased likelihood of hemorrhage in the event of excessive per-fusion pressure, and iii) high oxidative metabolic demands ofseveral midline nuclei (oculomotor and hypoglossal) possiblyrendering the neuronal complement of these nuclei more sensi-tive to hypoxia in the event of fetal hypoperfusion.

FIG. 9. Diagrammatic representation of the fetal rat brainstem atapproximately E14 to E16, illustrating the position of the medianavascular (A) and proposed paramedian watershed zones (B) in themidbrain (above), isthmus and pontomedullary junction (below).This age of the fetal rat is approximately equivalent to the earlysecond trimester human fetus. Regions marked C are the well-vascularized lateral parts of the brainstem. 4, trochlear nucleus; 7n,facial nerve internal genu; 6, abducens nucleus; Aq, cerebralaqueduct; 4V, fourth ventricle.

FIG. 8. Cytochrome oxidase histochemistry of the brainstem. (A, B)Low- and high-power views of the mesencephalon at E12, showingdenser staining at the ventricular and pial surfaces of the neuroepithelium(B), particularly in the subpial region of paramedian zone (curvedarrow), where the somatic efferent nuclear column is emerging. Thehigh power view of cytochrome oxidase staining at E12 also showspunctate labelling. (C) The myelencephalon at E12, showing elevatedactivity in the region of the somatic efferent nuclear column(arrowhead, hypoglossal nucleus). (D) At E15, high activity is presentin the hypoglossal nucleus (XII), but activity is low in the midlineraphe region. Magnifications: (A, C) 123; (B) 963; (D) 243.


ACKNOWLEDGEMENTS

We would like to thank Professor Robert Osborne for makingthe brain stem of the infant with Möbius syndrome available to us.

We would also like to thank Mr. Collin Yeo for advice and assistancewith the electronmicroscopy, and Dr Luan-ling Zhang for technicalassistance.

REFERENCES

1. Ashwell, K. W. S.: The development of microglia in the albinorabbit retina. J. Comp. Neurol. 287:286–301; 1989.

2. Bär, T. H.; Wolff, J. R.: The formation of capillary basementmembranes during internal vascularisation of the rat’s cerebralcortex. Z. Zellforsch. 133:231–248; 1972.

3. Bobryshev, Y.; Ashwell, K. W. S.: Activation of microglia in hem-orrhage microzones in the human embryonic cortex: An ultra-structural description. Pathol. Res. Practice 192:260–270; 1996.

4. Bouwes Bavinck, J. N.; Weaver, D. D.: Subclavian artery disruptionsequence: Hypothesis of a vascular aetiology for Poland, Klippel-Feil and Möbius anomalies. Am. J. Med. Gen. 23:903–918; 1986.

5. Caley, D. W.; Maxwell, D. S.: Development of blood vessels andextravascular space during postnatal maturation of the cerebralcortex. J. Comp. Neurol. 138:31–48; 1970.

6. D’Cruz, O. F.; Swisher, C. N.; Jaradeh, S.; Tang, T.; Konkol, R. J.:Möbius syndrome: Evidence for a vascular aetiology. J. ChildNeurol. 8:260–265; 1993.

7. Dor, P.; Salamon, G.: The arterioles and capillaries of the brainstem and cerebellum: A microangiographic study. Neuroradiol-ogy 1:27–29; 1970.

8. Federman, R.; Stoopack, J. C. Moebius syndrome. J. Oral Surg.33:676–678; 1975.

9. Foix, C. H.; Hillemand, P.: Les artéres de l’axis encéphaliquejusqu’au diencéphale inclusivement. Rev. Neurol. 32:705–739; 1925.

10. Franklin, J. B.; Brent, R. L.: The effect of uterine vessel clampingon the development of rat embryos three to fourteen days old. J.Morphol. 115:237–290; 1964.

11. Fujita, I.; Koyanagi, T.; Kukita, J.; Yamashita, H.; Minami, T.;Nakano, H.; Ueda, K.: Möbius syndrome with central hypoventi-lation and brainstem calcification: A case report. Eur. J. Pediatr.150:582–583; 1991.

12. Gilles, F. H.: Hypotensive brain stem necrosis. Selective symmet-rical necrosis of tegmental neuronal aggregates following cardiacarrest. Arch. Pathol. 88:32–41; 1969.

13. Goddard, J.; Lewis, R. M.; Alcala, H.; Zeller, R. S.: Intraventricu-lar hemorrhage—an animal model. Biol. Neonate 37:39–42; 1980.

14. Goldberg, R. N.; Chung, D.; Goldman, S. L.; Bancalari, E.: Theassociation of rapid volume expansion and intraventricular hem-orrhage in the preterm infant. J. Paediatr. 96:1060–1063; 1980.

15. Goldstein, G. W.; Betz, A. L.: Recent advances in understandingbrain capillary function. Ann. Neurol. 14:389–395; 1983.

16. Hoyme, H. E.; Jones, K. L.; Dixon, S. D.; Jewett, T.; Hanson, J. W.;Bobinson, L. K.; Msall, M. E.; Allanson, J. E.: Prenatal cocaineexposure and fetal vascular disruption. Pediatrics 85:743–747; 1990.

17. Kankirawatana, P.; Tennison, M. B.; D’Cruz, O.; Greenwood,R. S.: Möbius syndrome in an infant exposed to cocaine in utero.Pediatr. Neurol. 9:71–72; 1993.

18. Leist, K. H.; Grauwiler, J.: Fetal pathology in rats following uterinevessel clamping on day 14 of gestation. Teratology 10:55–68; 1974.

19. Lipson, A. H.; Webster, W. S.; Brown-Woodman, P. D. C.; Osborn,R. A. Möbius syndrome: Animal model-human correlations andevidence for a vascular etiology. Teratology 40:339–350; 1989.

20. Ment, L. R.; Stewart, W. B.; Duncan, C. C.; Scott, D. T.; Lam-brecht, R. L.: Beagle puppy model of intraventricular hemor-rhage: Effect of indomethacin on cerebral blood flow. J.Neurosurg. 58:857–862; 1983.

21. Misztal, D. R.: The effects of prenatal cytotoxic brain damage onthe vasculature of the fetal cortex. BSc (Hons) thesis, UNSW; 1993.

22. Paxinos, G.; Ashwell, K. W. S.; Törk, I.: Atlas of the developingrat nervous system. San Diego: Academic; 1994.

23. Paxinos, G.; Huang, X-F.: Atlas of the human brainstem. SanDiego: Academic; 1995.

24. Risau, W.; Hallmann, R.; Albrecht, P.; Henke-Fahle, S.: Braininduces the expression of an early cell surface marker for blood–brain barrier specific endothelium. EMBO J. 5:3179–3183; 1986.

25. Risau, W.; Wolburg, H.: Development of the blood–brain barrier.Trends Neurosci. 13:174–178; 1990.

26. St. Charles, S.; DiMario, F. J., Jr.; Grunnet, M. L.: Möbius syn-drome: Further in vivo support for the subclavian artery disrup-tion sequence. Am. J. Med. Gen. 47:289–293; 1993.

27. Stewart, P. A.; Hayakawa, K.: Early ultrastructural changes in theblood–brain barrier vessels of the rat embryo. Dev. Brain Res.78:25–34; 1994.

28. Stopford, J. S.: The arteries of the pons and medulla. J. Anat.Physiol. 50:131–141; 1915.

29. Takashima, S.; Armstrong, D.; Becker, L.; Bryan, C.: Cerebralhypoperfusion in the sudden infant death syndrome? Brainstemgliosis and vasculature. Ann. Neurol. 4:257–262; 1978.

30. Thakkar. N.; O’Neill, W.; Duvally, J.; Liu, C.; Ambler, M.:Möbius syndrome due to brainstem tegmental necrosis. Arch.Neurol. 34:124–126; 1977.

31. Webster, W. S.; Lipson, A. H.; Brown-Woodman, P. D. C.;Osborn, R. A.: Möbius unmasked: Pathogenesis of the Möbiussyndrome in an animal model. Teratology 38:199; 1988.

32. Webster, W. S.; Lipson, A. H.; Brown-Woodman, P. D. C.: Uter-ine trauma and limb defects. Teratology 35:253–260; 1987.

33. Webster, W. S.; Brown-Woodman, P. D. C. Cocaine as a cause ofcongenital malformations of vascular origin: Experimental evi-dence in the rat. Teratology 41:689–697; 1990.

34. Webster, W. S.; Brown-Woodman, P. D. C.; Lipson, A. H.;Ritchie, H. E.: Fetal brain damage in the rat following prenatalexposure to cocaine. Neurotoxicol. Teratol. 13:621–626; 1991.

35. Wong-Riley, M.; Merzenich, M. M.; Leake, P. A.: Changes inendogenous reactivity to DAB induced by neuronal activity.Brain Res. 141:185–192; 1978.

36. Yoshida, Y.; Yamada, M.: Endothelial fenestrae in the rat fetalcerebrum. Dev. Brain Res. 44:211–219; 1988.

Documents

Is There a Zone of Vascular Vulnerability in the Fetal Brain Stem?