Work Unit 127- 49- 02- 44-21

MORPHOLOGICAL STUDIES OF LAMINAR IJ3SIONS

Webb Haymaker

fhes Research Center, NASA

Moffett Field, California

(For Dr. John Lawrence, Donner Laboratory and Donner Pavilion, Berkeley,

California, as one of a series of essays for use at the Meeting of AEC

Biomedical Advisory Committee t o be held in Berkeley, March 10 and 11,

1967 * 1

MORPHOLOGICAL STUDIES OF LAMINAR LESIONS

Webb Haymaker

Ames Research Center, NASA

Moffett Field, California

A s Jerzy Rose once related, the first e f fo r t t o produce a laminar

lesion had behind it a neurophysiological objective.

of the six laminae of the cerebral cortex could be destroyed, leaving

the others intact , then it might be possible t o assess the contributions

made by the various laminae t o cortical. e l ec t r i ca l act ivi ty , as recorded

from the cerebral surface. So the first experiment w a s done t o see i f a

lamina could be selectively destroyed. This pioneer study, reported

10 years ago (1957) by Malis, Loevinger, Kruger and Rose," proved tha t

the approach w a s feasible. In two cats i n which a 5 x 5 m area of the

cerebrum w a s exposed t o 10-MeV proton radiatidn they demonstrated that a

longitudinal zone of the cortex 0.8 mm deep to the cerebral surface

could be severely damaged without materially injuring the intervening

tissue. In stained sections the damaged zone was found t o have a sharp

lower border and an uneven upper border, and i t s width was 40 to 100 p,

depending on dosage. Since t issue shririks about 35% during processing,

the actual width of the band would be correspondingly greater. To th i s

"Bragg-peak zone, '' o r "band, " they gave the misnomer, "laminar '' lesion,

a termwe sha l l probably have t o l i v e with.

If one o r another

I n actual i ty the zone w a s

pseudolaminar, as would be expected from borribardment with a mnoenergetic

beam. Nonetheless, the lesions were precisely enough situated t o serve

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as a wholely satisfactory mdel for the neurophysiological purposes

intended. Some results along thia line are availableOx5

Rose and his associate^,^" in following up that first study in cats, decided that the initial step should be a careful histological

study. They used rabbits and concentrated on silver methods that

specifically impregnate nerve cells and their processes. As in the

cats, the "Bragg-peak band" of damage in the rabbits was peculiar in

that only nerve cells were destroyed, leaving intact the rest of the

tissue elements, which included glia and fiber processes. From this

study came the remarkable observation that new axons grew into the

cell-depleted band. That nerve fibers in the brain can "regenerate"

had long been known, but here in the laminar preparation was a model

in which the phenomenon was open to study.

is sectioned, as many as 40 new axons sprout from a single nerve fiber

in the proximal stwtorp, and sprouting of much the same magnitude seemd

a l s o to be occurring in the laminar preparation.

however, not entirely apt, as no proof was provided that the particle

radiation amgutated a sizable n W e r of the fibers in their course

vertically through the band.

matrix in the band served as a latticework on which the new axons could

clirnb.

in that a semblance of the previous fiber-architecture was achieved,

but usually not.

When a peripheral nerve

The comparison is,

Be that as it my, the persisting tissue

There was sometines an orderliness in the regrowth in the band

A subsequent study, by Krm@er and Clemente,'? confirmed all this,

and showed that new mons could grow out not only in the cat and rabbit,

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but also i n the rat and monkey.

observed tha t axonal growth occurred not only i n the cerebral cortex

but a lso i n the cerebellar cortex.

a t both s i t e s tha t a f t e r a few months the axons could easi ly be seen i n

stained sections with the unaided eye.

the band was evident on about the 18th day postexposure.

into the band from &..long distance away, i.e., from a region of the white

matter near the corpus callosum. Moreover, it w a s shown tha t the new

axons i n the band were being surrounded by a sheath of myelin, but the

mechanism by which th i s occurred was obscure.

t o be requis i te to myelin formation, i n the sense that the myelin layers

encircling axons are an extension of the oligodendroglial cytoplasm.

reason fo r mentioning a l l t h i s i s t o indicate tha t -- i n vivo models are

needed if one i s to get a t the heart of the problem of how, for example,

remyelination i s achieved under some conditions of CNS injury, 'jX3 but

not under other conditions, such as rmiLtiple sclerosis. The laminar

preparation i s admirably suited as such a model.

Further, i n the r a t Estable-Puig e t &e5

Such a heavy growth of axons occurred

Entrance of the new axons in to

Many grew upward

Oligodendroglia appear

One

Speaking of possible applications of the laminar model t o c l in i ca l

problems i n man, we wonder whether, with the use of the mdel, nerve

f ibers could be induced to grow more effect ively up and down the spinal

cord i n paraplegics,

cord to par t ic le radiation i n the acute stage of injury create a l a t t i c e -

work on which new axons could more effectively grow?

It i s a w i l d idea, but could not exposure of the

Studies on laminar preparations were undertaken i n the ear ly days

without any idea as t o where they might lead. W e used t o come at times

from Washington t o work with D r . Tobias and h i s associates. I would put

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brain material in to a l l kinds of fixatives, then, on coming hom,

s t a in sections i n various ways.

of the NIH, came along and brought Rossmanss fluid, which contains a

number of . f ix ing ingredients.

f luid.

pretreatment with a-amylase he found that the astrogl ia i n the irra-

diated area (but not i n the control areas) contained par t ic les of

glycogen.'"

l eve l (45-70 mg per LOO gm of t issue i n the rat) usually not detectable

with use of the PAS stain. Only on considerable increase does the

glycogen become evident i n sections, and then i n the form of granules,

which are stained red by PAS. It was appropriate that the glycogen

increase should occur i n the astrocyte, for carbohydrate metabolism

i n th i s c e l l i s probably very intense. Through i t s cytoplasmic processes

wrapped around vessels, t h i s c e l l also has transport functions, carrying,

for example, glucose from the blood stream and discharging it into the

t issue, for use by such elements as nerve cel ls .

On one of these t r i p s Igor matzo,

He put some of the brain blocks i n t h i s

On staining the sections by the PAS method with and without

Glycogen i s normally present i n the brain, but i s a t a

matzo, and shortly thereafter Mlquel e t a1.,19,21 observed that the

glucose coming from the blood stream into the irradiated par t of the brain

was not being used properly and w a s being stored as glycogen. This obser-

vation was a breakthrough, i n tha t a dependable means was now available

for the study of CNS energy metabolism. Essentially the glycogen a c c m -

l a t ion represented a "biochemical lesion," the intensi ty of which reached

a peak i n 48 hours, then declined over the next week o r two, when normality

w a s reached.

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The glycogen increase raised a11 kinds of questions as t o i t s

genesis. A favored view was tha t the radiation d i rec t ly damaged the

t issue such that inhibition of aerobic metabolism occurred, with the

resu l t that glucose i n the t issue was converted to glycogen - i n other

words tha t the accumulation was the outcome of a profound reduction i n

oxydative metabolism. This view found support i n an observation made

by S n e ~ h k o , ~ ~ i n an experiment i n rabbits i n which determinations were

made of f ree 0, content i n cerebral t issue following X-irradiation of

the head a t 1-3 kR. O2 tension in the t issue f e l l transiently i n some

of the animals. However, i n a l l the animals the tension soon increased

drast ical ly (by 7O-lOO$).

O2 concentration fluctuated in a wave-like manner, and remained above

the 70% increase level.

and thus it was concluded that the r i s e i n O2 concentration i n the

brain was indicative of lowered oxidative processes. There seem no

bet ter explanation of acutely developing glycogen increase.

Subsequently - for a t l ea s t 7 hours - the

No para l le l was found w i t h hemodynamic shifts ,

The problem of O2 tension i n the brain following i r radiat ion is ,

however, not that simple, as shown by Aleksandrovskayal i n a histology

study i n rats. The radiation conditions she used were, however, so

different from those i n the preceding experiment that comparison i s not

valid. The r a t s were to t a l ly irradiated i n fractional doses of 50 R

weekly fo r a maximum of 250 R, and they were sacrificed a t various

periods of time up to one mnth a f te r the last exposure. Pathological

changes favoring a hy-poxic.effect were laminar lesions i n the cerebral

cortex, destruction of nerve cel ls , and multiplication of oligodendroglia.

According to the author: "On taking into account the high oxygen .

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requirement of an organism exposed to the action of penetrating radiation

it can be assumed tha t the usual amount of oxygen supplied t o the brain i s

found to be inadequate and a de f i c i t develops i n the oxygen supply of the

brain, o r t o put it differently, a secondary anoxia develops."

visible changes i n vessels was considered t o support t h i s view. While

lack of visible vascular change does not necessarily mean tha t it did

not exis t , and while vascular change may have occurred in th i s experiment

and been pathogenic, nonetheless the viewpoint i s refreshing, and certainly

has some substance.

Lack of

A s if the O2 factor were not enough t o cope with i n evaluating t issue

damage, there i s s t i l l another factor to be reckoned with, and that i s

brain edema. Here, a hypoxic s t a t e of the t issue i s inevitably a

complication.

To evaluate the significance of an edematous process i n radiation

injury a model w a s needed.

than the laminar preparation, i n which, as stated, only the upper par t

of the cortex i s damaged. In studies on the rat and cak carried out with

the use of t h i s model, it w a s found2' that very quickly a f t e r 48-MeV

a-par t ic le i r radiat ion given i n a large dose (24 bad) edema f lu id began

t o permeate the white matter beneath the band, u n t i l i n a day o r two a l l

the white matter throughout the cerebrum w a s flooded by the fluid. But

the interes.ting point w a s that glycogen accumulated i n as t rogl ia through-

out a l l the cerebral cortex, even i n that far thest removed from the s i t e

of radiation injury (Miquel, unpublished).

equally far-flung glycogen disturbance occurred a f te r a very s m a l l stab

wound had been made i n the cortex (Miquel and Ibrahim, unpublished).

None could be found that had more advantages

Then it was found that an

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This raised questions which have not as yet been answered.

reflexly-induced edematous process not visible under the light microscope

be held accountable? Increased glycogen content in the cortex must

necessarily go hand-in-hand with a reduction in brain function. Indeed,

?6ivdnek1* found a correlation be tween duration of glycogen accumulation

(following application of strychine to the cortex, which should have

Could some

induced an edematous state) and duration of abnormal conditioned reflexes.

In this connection, Miquel and Haymaker” observed in the totally irra-

diated brain (of the rat) that the largest accumulation of glycogen was in

a structure heavily concerned in emotive functioning, i.e., the hippo-

campus. The future requires that more consideration be given t o these

wider implications of laminar and other radiation-induced lesions. A

more sensitive means of glycogen detection is needed, as the glycogen

response about which we have been talking is not detectable as doses

below 1 bad. Perhaps further studies f o r the detection of glycogen

biochemically, by a method already established for laminar preparations,26

will prove profitable.

This gives something of the story of the investigations that were

opened up in our own laboratory by the chance observation that particle

irradiation resulted in glycogen accumulation.

This discussion is not supposed to exceed a certain length, and

since something needs to be said about the nerve cell, little can be

said about other matters. a-particle radiation in a dose of 12 bad

(surface dose) invariably*results in 2 o r 3 days in the appearance of

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mitotic figures at the s i t e s of neuroglia.

neuroglia do not divide mitoticaJ.lyJ

vascular permeability disturbances occurring i n the laminar preparation

can pers i s t f o r as long as 13 days (as shown with the use of fluorescein-

labelled albumin [FIJA],~,”,’~) and prominent vascular changes, as viewed

electron microscopically, can l a s t some 22 days.17 Thus, vascular a l t e r -

ations during such t i m e periods can be a source of parenchymal injwry.

Vessels i n irradiated t issue can be functionally defective even though

mrphologically they appear perfectly normal, o r pract ical ly ~ 0 . ~ 9 ~ ~

In this connection, any c l a i m tha t ionizing radiation primarily destroys

nerve ce l l s - say a f t e r a latency of some days o r weeks - shouLd be

considered unfounded d e s s backed up by vascular permeability studies.

However, som say tha t

A t about 6 bad (surface dose)

The oligodendrocyte has such a refined digestion system tha t mesenchymal

ce l l s may not be called into the laminar preparations t o clean up the

debris.18

detection of CNS radiation damage, the paper by Neumayr and Thurnher22

should not be neglected,

Lastly, i f one i s looking f o r a sensit ive system f o r the

I n regard t o nerve cel ls , some people might think tha t the brain is

But on ly about 1/10 the c e l l s a r e nerve composed w s t l y of these ce l l s ,

ce l l s ,

lial c e l l s and astrocytes, t o keep a nerve c e l l going, The nerve c e l l

i s thought t o be p a r t i c d a r l y radioresistant because it i s not subject

t o divisional processes which make other ce l l s radiosensitive; the nerve

c e l l uses prac t ica l ly aJ.1 i t s energy i n communication. The defenses set

It thus takes an average of 10 cel ls , such as vascular endothe-

up fo r the preservation of the in tegr i ty of the nerve ce l l s suff ice when

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the en t i re head i s irradiated, as under these conditions the nerve c e l l

can go uninjured following exposure t o 1000's of rad, yet oligodendroglia

not far away w i l l be destroyed by 200 rad2," (p r lmr i ly? secondarily?).

However, despite clll the defenses'available, the nerve c e l l i s the mst

vulnerable element when the radiation f i e l d i s very small, such as i n

the laminar preparation.

context i s tha t while vascular ce l l s and glial ce l l s react act ively to

the radiation injury and pers is t , the nerve c e l l dies.

What i s meant by "most vulnerable" i n t h i s

There seems t o be a discrepancy i n the dosage needed t o wipe out a

narrow band of nerve cells.

that a 1.5 krad surface dose of 48-MeV a-par t ic les (7.5 krad, peak dose)

w a s suff ic ient t o destroy a very narrow band of nerve cells - say a band

25 IJ. thick - i n 7 months.

1 x lo8 a/c$-sec, and dose r a t e 10 krad/min.

I n one experiment, Janssen e t al.li found

Par t ic le flux i n t h i s experiment w a s

On the other hand, Zeman

e t

an aperture 25 p i n diameter, found no discernible damage i n 6-1/2 months

a f t e r exposure t o 225 krad (10.7 x lo9 d/cni?-see; 8 krad/sec), though

nerve-cell destruction did occur i n 24 days when the dose was doubled.

The need f o r massive dosage t o destroy nerve ce l l s i n a 25 p, track was

i n bolribarding the brains of mice with 22.5-MeV deuterons through

subsequently confirmed.27 How is one t o account for such wide differences

i n resul ts?

volume of brain i r radiated i n the Janssen e t al. experiment can account

f o r the difference.

People say it must be a matter of geometry, tha t the greater

More concrete information i s needed. Although much is known i n

regard t o dose-tissue volume factors i n pathogenesis when larger areas of

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the brain are irradiated, much s t i l l remains t o be learned of the factors

involved i n damage incurred i n t i ny brain areas.

do i n collaboration with D r . Tobias and h i s group i n Berkeley i s t o bordbard

many 25 IJ. f i e lds i n the same brain with protons or a-par t ic les i n an e f fo r t

t o s e t t l e t h i s problem.

t o spacing of the par t ic le tracks. This would be, as it were, a f irst

approximation t o a study of the effects of heavy primary primaries once

the presently planned accelerator becomes available.

What we are hoping t o

Tolerance doses would be established i n re la t ion

I n th i s connection, there is the problem of the IiBE of par t ic le

radiation. For most endpoints the RBE has been shown t o be close t o 1.

But i n regard t o the brain it seem l ike ly tha t the RBE of par t ic le radi-

at ion i s higher than l2 at l e a s t fo r cer ta in par t ic le energies. I n the

wnkey, 6 krad proton radiation (55 MeV) given a t 2 krad/min caused far

greater damage i n the same period (unpublished) than 30 krad

(1.2-1.4 MeV) given at 1 k.R/~nin.’ The comparison seems to be valid.

Also it has been found tha t 138-~ev protons had l e s s of an e f fec t on

the brain than 55-MeV or 32-MeV protons (unpublished).

y rays

A s t o the e f fec t

of par t ic les of different nuclear charge on the brain, we once ran an

experiment with Dr . Tobias i n which laminar lesions were made both with

protons and a-par t ic les of equal energy per nucleon (12 M~v) , ami given

i n the same dose and at the same dose rate. According t o the published

results, the width of the band w a s approximately the same i n both series.”

But there remains the hankering suspicion tha t the experiment was not well

enough designed t o provide a def ini t ive answer. Repetition i s therefore

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i n order.

accelerated larger nuclei become available, and thus t o get some idea

of the RBE of galactic cosmic rays so far as the brain i s concerned,

then considerably m r e work is needed with par t ic les now available.

If we are t o be ready t o do the crucial studies when

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'

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Res-ponse of the Nervous

System t o Ionizing Radiation.

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1.7

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