JOURNAL OF PATHOLOGY, VOL.

178: 182-189 (1996)

ASSESSMENT OF THE DISTRIBUTION OF

MITOCHONDRIAL RIBOSOMAL RNA IN MELAS AND
IN THROMBOTIC CEREBRAL INFARCTS BY IN SITU
HYBRIDIZATION
SETH LOVE AND DAVID A. HILTON

Depurtment of Neuropathology, Frenchay Hospital, Bristol BS16 ILE, U.K.

SUMMARY
In situ hybridization to mitochondrial ribosomal RNA (rRNA) has been used to study the distribution of mitochondria in
paraffin-embedded autopsy brain tissue from two patients with MELAS (mitochondrial myopathy , encephalopathy, lactic acidosis, and
stroke-like episodes) and other organs from one of the patients. Comparison of in situ hybridization and electron microscopic findings
in an antemortem biopsy specimen of pylorus from the latter patient showed a close correspondence between the distribution of
hybridization signal on light microscopy and of mitochondria in ultrathin sections. Strong hybridization signal was present over smooth
muscle fibres of the muscularis externa, which contained abnormal accumulations of mitochondria on electron microscopy. Hybridization
to sections of skeletal muscle confirmed previous reports of ‘ragged-red’ fibres in this disorder and of mitochondrial accumulations in the
walls of intramuscular blood vessels. To try to elucidate the role of vessel wall accumulation of mitochondria in the genesis of the
stroke-like lesions, the distribution of mitochondrial rRNA was assessed in sections of brain from both of the cases of MELAS and
several cases of atherothrombotic cerebrovascular disease. Blood vessels in and adjacent to the cerebral lesions of MELAS showed
strong hybridization signal with the mitochondrial probes, as was also seen in infarcts of various ages in the control brains. Only weak
signal was present in the walls of blood vessels distant from the lesions, in both MELAS and control brains. These findings suggest that
mitochondria accumulate in vascular endothelium and tunica media as a normal response to cerebral infarction or ischaemia. The
accumulation of mitochondria in the cerebral lesions of MELAS may, at least in part, be a reaction to the destructive effects of the
underlying metabolic dysfunction.
KEY WORDS-MELAS; in situ hybridization; mitochondrial rRNA; blood vessels; pylorus; skeletal muscle; pancreatic islets; cerebral
infarcts

INTRODUCTION The pathogenesis of the stroke-like episodes and other

MELAS (mitochondrial myopathy, lactic acidosis,
and stroke-like episodes) is an uncommon disorder of
children and young adults. Clinical features include
sudden onset of headaches, vomiting, convulsions,
cortical blindness, and hemiplegia or hemianopia.
Although there is usually some recovery between epi-
sodes, the natural history is of increasing clinical impair-
ment and, eventually, death. In most cases, there is a
single point mutation in mitochondrial DNA (mtDNA),
usually at nucleotide 3243, in the tRNAL“U(URR’ gene,2
although mutations at other positions, particularly
nucleotide 3271, have been detected in some MELAS
patients.’ The same nucleotide 3243 mutation has been
found in a subgroup of patients with maternally-
inherited diabetes mellit~s,~-’ usually in association with
sensorineural deafness, and we have also detected the
nucleotide 3243 mutation in association with chronic
intestinal pseudo-obstruction,8 known by the acronym
CIP0,9 although other acronyms have been applied
to this disorder including POLIP-polyneuropathy,
manifestations of MELAS and these allied mitochon-
drial disorders remains unclear. A higher proportion
of mtDNA is mutated in clinically affected than in
asymptomatic members of families with MELAS12 and
in some families, the age of onset seems to be inversely
related to the ratio of mutant to wild-type mtDNA.”
However, although some studies have found a correla-
tion between the relative amount of mtDNA in different
tissues and the clinical presentati~n,’~’’ in other studies
no such correlation has been demonstrable.83’‘-18 Sev-
era1 authors have raised the possibility that the lesions in
MELAS are essentially ischaemic in nature, due to a
‘mitochondrial angiopathy’.’’ 2’ Pathological studies of
the central nervous system have shown infdrct-like
lesions of varying ages, particularly in the cerebral
hemispheres, within which the occipital lobes are often
worst affected. There is evidence of increased activity of
the mitochondrial enzyme succinate dehydrogenase in
the intramuscular blood vessels of MELAS patients2*
and an increase in the amount of mtDNA was detected
by in situ h y b r i d i ~ a t i o n Some,
.~~ although not
1y320,24325

ophthalmoplegia, leukoencephalopathy and intestinal studies have reported abnormal accumulation of

pseudo-obstruction,” and MNGIE-mitochondria1 mitochondria within the endothelial cells and tunica
neurogastrointestinal encephalomyopathy . ’’ media of blood vessels in the brain in MELAS. These
observations have prompted speculation that the mito-
Addressee for correspondence: S. Love, Department of Neuro- chondrial accumulations may narrow the lumina of
pathology, Frenchay Hospital, Bristol BS16 ILE, U.K. arterioles sufficiently to cause ischaemia.
CCC 0022-34 17/96/020182-08 Received 8 February 1995
0 1996 by John Wiley & Sons, Ltd. Accepted 26 June 1995
 ISH TO MITOCHONDRIA IN MELAS AND CEREBRAL INFARCTS 183

To investigate the distribution of mitochondria within

the brains and other tissues of MELAS cases, we have
used in situ hybridization (ISH) with digoxigenin-
labelled probes to mitochondrial ribosomal RNA
(rRNA). We have previously shown this technique to
provide a good indication of the distribution of mito-
chondria, even in paraffin sections.27 To evaluate the
significance of the findings in the central nervous system,
we have also assessed the distribution of mitochondria
in cases of cerebral infarction due to atherothrombotic
cerebrovascular disease.

MATERIALS AND METHODS

Two patients with MELAS were studied. The clinical

and pathological findings in both patients have been
reported in detail previously26and the nucleotide 3243
mutation in mtDNA was demonstrated in autopsy
material from one of these patients by the olymerase
chain reaction (PCR) and Pan digestion.' ISH was
performed on paraffin sections of brain from both
patients and on the following additional material from
the patient with the nucleotide 3243 mutation: sections
of a pyloric biopsy that had been taken during life and Fig. 2-Strong ISH signal for mitochondrial rRNA in smooth muscle
sections of skeletal muscle, pancreas, liver, heart, and fibres of the muscularis externa, blood vessels (arrows), and myenteric
ganglia (inset) in a biopsy from a MELAS patient
kidney obtained at autopsy.
ISH was also performed on paraffin sections of several
thrombotic cerebral infarcts. The infarcts ranged in
age from a few days to months or longer. The sections
were obtained from a range of autopsy brain specimens Sections of appropriate normal control tissues-
and, in three cases, cerebral biopsies, from patients brain, pylorus, skeletal muscle, pancreas, liver, heart, or
without any clinical or biochemical features to suggest kidney-were included in each batch of ISHs.
mitochondrial disease.
Probes
Synthetic oligonucleotides, complementary to the 16s
subunit of mitochondrial rRNA and labelled at the 3'
end with poly-dUTP- 1 1-digoxigenin, were used as
described p r e v i ~ u s l y . ~ ~ - ~ ~

Fig. l-Section of a pyloric biopsy from a MELAS patient, showing
normal-looking antral mucosa, submucosa, and part of the muscularis
externa. Haematoxylin and eosin
In situ hybridization
Paraffin sections, 5 pm in thickness, were collected
onto aminopropyltriethoxysilane-coated glass slides.
The sections were dewaxed in xylene and alcohol,
washed in 2 x SSC at 70°C for 10 min, digested in
15 puglml proteinase K for 60 min at 37"C, and refixed in
0.4 per cent paraformaldehyde in 0.1 M phosphate
buffer for 20 min at 4°C. Hybridization was overnight
at 37°C in 5Opl of buffer containing 0.1 ndpl probe,
30 per cent formamide, 600 mM NaC1, 0.1 M phosphate
buffer, 10 per cent dextran sulphate, and 150pglml
sheared salmon sperm DNA. The sections were
washed in 2 x SSCl30 per cent formamide at 37T,
incubated for 10 min in Tris-HC1 (pH 7.5)/0.5 M NaC1/3
per cent bovine serum albumin, and then for 30 min
with anti-digoxigenin antibody conjugated to alkaline
phosphatase (Boehringer Mannheim). Bound anti-
body was visualized by reaction with nitroblue tetrazo-
lium salt and 5-bromo-4-chloro-3-indolyl phosphate
solution.
184 S. LOVE AND D. A. HILTON

Fig. .?-Electron micrograph showing smooth muscle fibres in the pyloric biopsy from the MELAS patient. The fibres contain
abnormal accumulations of mitochondria, including several that are of abnormal size and shape (large arrows) and a few with
osmiophilic inclusions (small arrow)

RESULTS glutaraldehyde-fixed tissue from the same biopsy. This

Pylovus
In sections stained with haematoxylin and eosin, the
biopsies appeared entirely normal (Fig. 1). On ISH,
strong signal was present in the mucosal epithelium, in
the walls of blood vessels, in submucosal and myenteric
ganglia, and in the smooth muscle of the muscularis
externa (Fig. 2). Hybridization to control sections of
normal pylorus showed strong signal in the mucosa,
blood vessels, and ganglia, but only weak signal in the
muscularis externa.
The distribution of signal on ISH was compared
with that of mitochondria in ultrathin sections of
confirmed the presence of numerous mitochondria in
smooth muscle fibres of the muscularis externa (Fig. 3),
also in ganglion cells, Schwann cells, and, to a lesser
extent, endothelial cells and cells in the tunica media of
intramuscular and submucosal blood vessels. Many of
the mitochondria were abnormally large or excessively
elongated and a few contained dense, osmiophilic
bodies. No paracrystalline inclusions were noted.
Skeletal muscle
There was no discernible hybridization to muscle
fibres in control sections and only scanty, variable
 1SH TO MITOCHONDRIA IN MELAS AND CEREBRAL INFARCTS
Fig. &Section of skeletal muscle from the MELAS patient hybridized
with probes to mitochondrial rRNA. The fascicle towards the top left
of the figure includes several fibres with abnormally strong hybridiza-
tion signal, including some in which the signal is predominantly
subsarcolemmal (small arrows). Note the strong hybridization to
interfascicular blood vessels (large arrows)
hybridization to blood vessels, Sections from the
MELAS patient included many fibres with excessive
hybridization signal (Fig. 4). In several of these, the
signal was strongest in the subsarcolemmal region, as
would be expected in mitochondrial myopathy. Most
interfascicular arterioles and venules, and some endo-
mysial capillaries, also showed strong hybridization for
mitochondrial rRNA. In arterioles, the hybridization
was to the tunica media as well as to the endothelium.
Pancreas
The pattern of hybridization to exocrine pancreatic
tissue in the MELAS patient was similar to that in
control sections, with moderate but quite variable signal
over acinar cells and ductular epithelium. Control tissue
included many pancreatic islets, which hybridized rela-
tively strongly with the mitochondrial probes. In the
MELAS patient, only two islets were identified in a
cross-section of pancreatic tail and these showed only
weak hybridization signal (Fig. 5).
Liver, heart, and kidney
No obvious abnormalities were noted in the pattern of
hybridization in the MELAS patient but tissue preser-
vation was suboptimal and the sections were unsuitable
for detailed assessment.
Brain
Blood vessels in and adjacent to the cerebral infarct-
like lesions of MELAS showed strong hybridization
185
Fig. 5-Sections of pancreas from the MELAS patient (a) and control
(b). Although the strength and pattern of mitochondrial ISH to
exocrine pancreatic tissue is similar in both sections, there is weaker
signal over the islet in the MELAS patient (arrow) than over the islets
in the normal control (arrows)
signal with the mitochondrial probes (Figs 6, 7, and 8).
This was a consistent finding in all the lesions examined
in both cases. The hybridization was to vascular
endothelium and tunica media and, in the more recent
lesions, to scattered macrophages. There was only weak
signal in the walls of blood vessels away from the
infarct-like lesions, although moderately intense hybridi-
zation signal was noted in scattered neurons, in the
larger blood vessels in the leptomeninges, and in choroid
plexus epithelium.
Thrombotic infarcts of all ages showed strong
hybridization to the walls of blood vessels (Figs 9 and
10) and, in recent lesions, to macrophages as well. As in
the cases of MELAS, there was also hybridization to
scattered neurons, to the larger blood vessels in the
leptomeninges, and to choroid plexus epithelium; there
was only weak hybridization to blood vessels at a
distance from the infarcts.
DISCUSSION
Our findings provide further evidence of the accumu-
lation of mitochondria in the walls of blood vessels in
some of the tissues affected by MELAS. The strong
hybridization to mitochondrial rRNA in the walls of
blood vessels in skeletal muscle is in keeping with the
observation by Hasegawa et of increased succinate
dehydrogenase activity and the detection of excessive
amounts of mtDNA by ISH.23 ISH also provided clear
evidence of the accumulation of mitochondria in the
186 S. LOVE AND D. A. HILTON
Fig 6-Pdrdkl sections of Lerebrdl cortex from d MELAS patient,
stained with haematoxylin and eosin (top) and hybridized for mito-
chondrid~rRNA (bottom) The cortex includes several old infarct-like
lesions. within which blood vessels show strong mitochondrial hybridi-
zation signal (arrous to corresponding lesions in haematoxylin and
eosiii-stnined section and ISH prepdrdtion)
endothelium and tunica media of blood vessels within
the cerebral lesions of MELAS.
The role and pathogenetic significance of the mito-
chondria] accumulations in the walls of blood vessels is,
however, debatable. Several observations argue against
the simple hypothesis that the mitochondrial aggre-
gates narrow the vascular lumina so much as to cause
Fig 7-Section through an old MELAS lesion, viewed at higher
magnification, showing much stronger hybridization for mitochondrid
in the walls of blood vessels within the lesion (towards the bottom of
the figure) than in those outside (top of figure, Arrows to some)
ischaemic damage. No angiographic abnormalities have
been noted in these patients.2’’26 Studies of cerebral
blood flow have shown flow to be increased rather than
decreased, both within the lesions and in the preserved
parts of the Our present observations suggest
that the accumulation of mitochondria in the walls of
blood vessels may in part be a reactive phenomenon, the
pattern closely resembling that in cerebral infarcts. In
both MELAS and thrombotic infarcts, the ISH signal
with the mitochondrial probes was much weaker away
from the lesions than within or adjacent to them.
The precise stimuli for mitochondrial proliferation are
not known but may include ischaemia, tissue acidosis,
and a variety of chemical mediators released during
tissue necrosis. Heffner and Barrod2 recorded striking
proliferation of intramuscular mitochondria in tibialis
anterior in the rat within 24 h of application of a rubber
band tourniquet to the thigh. The resulting accumula-
tions included many mitochondria that were abnormally
large or contained paracrystalline inclusion bodies.
Other reports of abnormalities in the distribution or
appearance of mitochondria in ischaemic skeletal muscle
include those of Reznik3’ and Karpati et In tissue
culture, mtDNA with the MELAS-associated mutation
has a marked replicative advantage over wild-type
mtDNA.35One might therefore expect that proliferation
of mitochondria in vivo would cause an increase in the
ratio of mutant to wild-type mtDNA but, a t least in
skeletal muscle fibres, this does not seem to be the case:
the relative proportions of mutant and wild-type
mtDNA as assessed by PCR seem to be the same in
‘ragged-red’ as in ‘non-ragged-red’ fibres.’6
 ISH TO MITOCHONDRIA IN MELAS AND CEREBRAL INFARCTS
Fig. &Parallel sections through the edge of a recent infarct-like lesion
in a MELAS patient. One section has been stained with haematoxylin
and eosin (top); the other has been hybridized for mitochondrial
rRNA (bottom). Blood vessels within and immediately adjacent to the
lesion (towards the bottom of the figures) show strong hybridization
signal
It is possible that the pathogenesis of the lesions in
MELAS differs in different tissues, as does the under-
lying metabolic defect. MullerHocker et nl.37 found
reduced levels of cytochrome c oxidase activity and
immunohistochemically-detectable protein in heart and
skeletal muscle, but not brain. Conversely, NADH
dehydrogenase (complex I) activity was markedly
187
Fig. 9-Parallel sections through the edge of a recent infarct in a
control brain. One section has been stained with haematoxylin and
eosin (top); the other has been hybridized for mitochondria1 rRNA
(bottom). As in Fig. 8, there is stronger hybridization signal over blood
vessels within and adjacent to the lesion (which is towards the bottom
of the figures) than over those further away (arrows to some)
reduced in brain (to 20 per cent) but only moderately
reduced (to 50 per cent) in skeletal muscle.
Little has previously been reported on the pathologi-
cal basis of the gastrointestinal abnormalities in
MELAS. Magnetic resonance imaging in patients
with the syndrome of polyneuropathy, ophthalmoplegia,
188 S. LOVE AND D. A. HlLTON
Fig. 10-Section through an old, atherothrombotic infarct it1 the
cerebral cortex of a control brain. Blood vessels within the infarct
show strong hybridization signal for mitochondrial rRNA, whereas
those further away are relatively weakly labelled (arrows to some)
leucoencephalopathy, and chronic intestinal pseudo-
obstruction was reported by Simon et a1." to show
'endoneurial fibrosis and demyelination' in the periph-
eral nervous system. These authors attributed the
gastrointestinal dysmotility to visceral neuropathy.
However, apart from our own brief previous descrip-
tion,26 there are, to our knowledge, no reports of the
pathology of the gastrointestinal tract itself. The pres-
ence of large accumulations of mitochondria in the
smooth muscle fibres of the muscularis externa are
similar to the findings in skeletal muscle and suggest that
the gastrointestinal dysmotility may be myogenic rather
than neurogenic in origin.
The association of the mtDNA nucleotide 3243
mutation with a subtype of diabetes mellitus is well
d o ~ u m e n t e d . ~ Most patients do not have other
features of MELAS, but Kadowaki et ~ 1 found . ~ bio-
chemical evidence of diabetes mellitus in 8 of 39 patients
with MELAS due to the nucleotide 3243 mutation. Only
limited conclusions can be drawn from our observations
on the pathology of the pancreas in a single case of
MELAS, particularly as a diagnosis of diabetes mellitus
was never suggested during life in this patient. The
paucity of islets and the reduced mitochondrial hybridi-
zation signal are, however, consistent with observations
of decreased secretion of insulin in diabetes due to the
nucleotide 3243 rnutation5x6 and it will be of interest to
see whether or not these observations hold true in other
patients with this mutation.
Finally, the close correlation between the findings on
ISH and on electron microscopy of the pyloric biopsy
confirm the validity of using ISH for histological detec-
tion of mitochondria. The present study illustrates how
the technique can be used to analyse the distribution of
mitochondria in a wide range of archival paraffin-
embedded tissues.
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