Pontocerebellar

hypoplasia

type

a

neuropathological

update

Pontocerebellar

hypoplasia

type

-LRB-

MIM

-RRB-

an

autosomal

recessive

neurodegeneration

with

fetal

onset

was

studied

in

six

autopsies

with

ages

at

death

ranging

between

and

years

Three

patients

were

distantly

related

A

case

of

olivopontocerebellar

hypoplasia

-LRB-

OPCH

MIM

-RRB-

was

studied

for

comparison

Typical

findings

are

short

cerebellar

folia

with

poor

branching

-LRB-

hypoplasia

-RRB-

relative

sparing

of

the

vermis

sharply

demarcated

areas

of

full

thickness

loss

of

cerebellar

cortex

probably

resulting

from

regression

at

an

early

stage

of

development

segmental

loss

of

dentate

nuclei

with

preserved

islands

and

reactive

changes

segmental

loss

in

the

inferior

olivary

nucleus

with

reactive

changes

loss

of

ventral

pontine

nuclei

with

near

absence

of

transverse

pontine

fibers

and

sparing

of

spinal

anterior

horn

cells

Variable

findings

are

cystic

cerebellar

degeneration

found

in

two

with

vascular

changes

limited

to

the

cerebellum

in

one

Comparison

to

olivopontocerebellar

hypoplasia

-LRB-

OPCH

-RRB-

strongly

suggests

a

continuum

of

pathology

between

this

disorder

and

Immunohistochemical

evaluation

of

the

endoplasmic

reticulum

stress

response

is

negative

We

conclude

that

the

neuropathological

findings

in

are

sufficiently

specific

to

enable

an

unequivocal

diagnosis

based

on

neuropathology

Introduction

The

term

pontocerebellar

hypoplasia

-LRB-

PCH

-RRB-

is

applied

to

a

group

of

autosomal

recessively

inherited

neurodegenerations

with

prenatal

onset

The

distinguishing

attribute

of

this

group

is

the

presence

of

combined

pontocerebellar

hypoplasia

and

atrophy

present

at

the

time

of

birth

Supratentorial

structures

are

affected

though

less

prominently

The

group

has

not

been

clarified

genetically

or

biochemically

An

initial

proposal

for

classification

-LSB-

-RSB-

is

based

on

two

types

in

which

type

-LRB-

-RRB-

associates

PCH

with

spinal

anterior

horn

cell

degeneration

-LSB-

-RSB-

while

type

-LRB-

-RRB-

associates

PCH

with

clinical

extrapyramidal

involvement

and

absence

of

anterior

horn

degeneration

-LSB-

-RSB-

Early

detailed

neuropathological

reports

on

PCH

-LSB-

-RSB-

in

retrospect

may

have

been

cases

of

but

can

not

be

matched

with

the

customary

classification

because

of

lack

of

clinical

details

Neuropathological

finding

in

-LSB-

-RSB-

is

microencephaly

with

severe

cerebellar

and

ventral

pontine

hypoplasia

Typical

pathological

findings

are

-LRB-

-RRB-

subtotal

loss

of

ventral

pontine

neurons

and

transverse

pontine

fibers

with

preservation

of

long

fiber

tracts

-LRB-

-RRB-

atrophy

and

hypoplasia

of

the

cerebellar

hemispheres

with

relative

sparing

of

the

vermis

and

flocculi

variable

loss

of

Purkinje

cells

and

internal

granule

cells

and

reduction

in

folial

length

-LRB-

-RRB-

patchy

loss

of

cerebellar

dentate

neurons

with

the

remaining

dentate

neurons

grouped

in

islands

-LRB-

-RRB-

loss

of

neurons

in

the

inferior

olivary

nucleus

with

the

winding

pattern

essentially

spared

-LRB-

-RRB-

absence

of

the

medullary

arcuate

nuclei

Findings

in

are

essentially

similar

but

preservation

of

spinal

anterior

horn

cells

distinguishes

from

-LSB-

-RSB-

Microscopic

supratentorial

findings

in

are

non-specific

with

neuronal

loss

in

various

compartments

including

the

cerebral

cortex

while

myelin

and

myelination

remain

unaffected

Ultrastructural

findings

in

a

single

reported

neocortical

biopsy

are

progressive

neuronal

loss

in

all

layers

and

a

peculiar

degeneration

in

neurons

in

all

layers

that

appear

to

start

with

patches

of

darkened

endoplasmic

reticulum

-LSB-

-RSB-

Diagnosis

during

life

is

based

on

a

combination

of

findings

on

MRI

-LRB-

Fig.

-RRB-

a

profile

of

clinical

neurological

deficits

that

includes

severe

cognitive

delay

swallowing

disturbance

and

chorea/dystonia

-LRB-

less

often

spasticity

-RRB-

exclusion

of

metabolic

and

chromosomal

disorders

with

a

similar

MRI

pattern

and

a

family

history

compatible

with

autosomal

recessive

inheritance

In

typical

cases

behavioral

and

motor

development

is

almost

stagnant

from

the

beginning

with

onset

of

chorea/dystonia

during

the

first

year

and

progressive

microcephaly

-LSB-

-RSB-

Differential

diagnosis

requires

exclusion

of

glycosylation

disorders

especially

congenital

disorder

of

glycosylation

type

-LRB-

-RRB-

-LSB-

-RSB-

and

Muscle-Eye-Brain

disease

-LSB-

-RSB-

sequelae

of

extreme

prematurity

-LSB-

-RSB-

and

chromosomal

disorders

-LSB-

-RSB-

Overlapping

neuropathological

features

exist

between

and

olivopontocerebellar

hypoplasia

-LRB-

OPCH

-RRB-

a

more

severe

disorder

with

an

essentially

similar

combination

of

hypoplasia

and

degeneration

affecting

the

hindbrain

-LSB-

-RSB-

Autosomal

recessive

inheritance

in

OPCH

is

suggested

by

its

recurrence

pattern

in

families

Clinical

findings

are

variable

but

include

such

indicators

of

prenatal

onset

as

polyhydramnios

and

contractures

Its

main

structural

features

are

severe

cerebellar

hypoplasia

with

emphasis

on

the

hemispheres

absence

of

neurons

in

the

ventral

pons

subtotal

absence

of

cerebellar

dentate

nuclei

diminished

or

absent

olivary

winding

and

absence

of

spinal

anterior

horn

involvement

In

a

recent

PCH

classification

the

original

types

and

have

remained

identical

while

olivopontocerebellar

hypoplasia

has

become

type

-LSB-

-RSB-

Type

with

optic

atrophy

as

its

main

distinguishing

clinical

feature

-LSB-

-RSB-

has

not

been

studied

neuropathologically

yet

while

type

until

now

is

represented

by

a

single

family

The

number

of

papers

addressing

the

neuropathology

of

with

sufficient

clinical

detail

to

support

a

type

classification

is

limited

-LSB-

-RSB-

and

results

of

immunohistochemistry

have

not

been

reported

yet

The

aim

of

the

present

report

is

to

provide

a

range

of

pathological

findings

based

on

a

series

of

six

autopsies

with

ages

at

death

varying

between

months

and

years

and

to

provide

an

update

on

neurodegenerative

features

using

more

specific

staining

techniques

Special

attention

is

given

to

variability

in

cerebellar

degeneration

degenerative

changes

in

pons

and

lower

brainstem

and

the

types

of

astroglial

and

microglial

reaction

Three

patients

-LRB-

no.

-RRB-

originate

from

an

area

with

a

high

degree

of

intermarriage

and

are

related

by

genealogy

A

case

of

olivopontocerebellar

hypoplasia

-LRB-

-RRB-

is

included

for

comparison

of

its

neuropathology

to

Fig.

of

patient

at

months

a

Midsagittal

inversion

recovery

sequence

flat

aspect

of

ventral

pons

and

moderate

vermal

hypoplasia

b

Coronal

image

severe

hypoplasia

of

the

cerebellar

hemispheres

-LRB-

arrows

-RRB-

together

filling

only

a

fraction

of

the

posterior

fossa

Materials

and

methods

Standard

autopsies

were

performed

on

all

patients

mentioned

except

patient

who

underwent

a

coroner

's

autopsy

with

only

the

cerebellum

and

brainstem

made

available

for

the

present

study

-LRB-

Table

-RRB-

Table

profiles

macroscopic

and

principal

microscopic

findingsPatient

no.

of

PCHPCH-2PCH-2PCH-2PCH-2PCH-2PCH-2OPCHParents

relatedNoNoYesYesYesUncertainYesNeonatal

periodPoor

swallowingPoor

swallowing

seizureUnrestSeizurePoor

swallowingPoor

swallowing

unrestAbsent

spontaneous

respiration

tremor/clonusHead

circumference

at

birth

SD

SD

SDMicrocephaly

progressive

after

birth

Motor

development

after

birthNo

motor

control

some

visual

pursuit

Minimal

head

control

some

visual

pursuitMinimal

head

control

some

visual

pursuitNo

motor

control

some

visual

pursuitNo

motor

control

no

visual

pursuitNo

motor

control

no

visual

pursuitChorea/dystonia

Spasticity

Forced

flexion

of

extremitiesSeizures

Previous

publicationPatient

-LSB-

-RSB-

Patient

-LSB-

-RSB-

Patient

-LSB-

-RSB-

Patient

-LSB-

-RSB-

Age

at

year

years

years

years

years

daysCause

of

deathCot

deathHyperthermiaCachexiaPneumoniaPneumoniaHyperthermia

rhabdomyolysisAbsent

spontaneous

respirationTotal

brain

weight

-LRB-

g

-RRB-

After

fixation

fixation

brain

weight

g

SD

of

cerebellum

Cerebellar

cortexSparse

segmental

lesions

in

hemispheres

diffuse

loss

of

P

and

granule

cells

Sparse

segmental

lesions

in

vermis

and

hemispheres

diffuse

loss

of

all

P

and

granule

cellsNear

total

destruction

only

GFAP

positive

outlines

remainingSegmental

lesions

in

hemispheres

diffuse

loss

of

all

P

and

granule

cellsSegmental

lesions

in

vermis

and

hemispheres

diffuse

loss

of

all

P

and

granule

cellsSegmental

lesions

in

vermis

and

hemispheres

diffuse

loss

of

all

P

and

granule

cellsMajor

-LRB-

dorsal

-RRB-

part

of

vermis

and

hemispheres

denuded

sparing

of

flocculus

and

nodulusDentate

nucleusIslandsIislandsNo

remnantsIslandsIslandsIslandsIslandsCerebellar

cysts

Inferior

olivary

nucleusReduced

folding

segmental

lesionsReduced

folding

segmental

lesionsTotal

loss

of

structureReduced

folding

segmental

lesionsReduced

folding

segmental

lesionsReduced

folding

segmental

lesionsNo

foldingVentral

ponsLoss

of

nuclei

transverse

fibersLoss

of

nuclei

transverse

fibersLoss

of

nuclei

transverse

fibersLoss

of

nuclei

transverse

fibersLoss

of

nuclei

transverse

fibersLoss

of

nuclei

transverse

fibersLoss

of

nuclei

transverse

fibersaRelated

by

common

ancestry

-LRB-

Fig.

-RRB-

bReference

for

fresh

brain

weights

-LSB-

-RSB-

cReference

for

fixed

brain

weights

-LSB-

-RSB-

Tissue

preparation

and

immunocytochemistry

Formalin-fixed

paraffin-embedded

tissue

was

sectioned

at

μm

and

mounted

on

organosilane-coated

slides

-LRB-

SIGMA

St

Louis

MO

USA

-RRB-

Representative

sections

of

all

specimens

were

processed

for

immunocytochemical

reactions

to

synaptophysin

-LRB-

polyclonal

rabbit

DAKO

-RRB-

microtubule-associated

protein

-LRB-

polyclonal

rabbit

Sigma

-RRB-

calbindin

-LRB-

mouse

clone

Sigma

-RRB-

-LRB-

Sternberger

Monocl.

IgG

-RRB-

phosphorylated

neurofilament

-LRB-

NF

mAb

clone

DAKO

-RRB-

myelin

basic

proteïn

-LRB-

MBP

polyclonal

Neomarkers/RB

-RRB-

vimentin

-LRB-

mouse

clone

DAKO

Denmark

-RRB-

glial

fibrillary

acidic

protein

-LRB-

GFAP

polyclonal

rabbit

DAKO

Denmark

-RRB-

-LRB-

HLA

-RRB-

DP

DQ

DR

-LRB-

mouse

clone

DAKO

Glostrup

-RRB-

HLA-DR

-LRB-

mouse

clone

Sigma

USA

-RRB-

-LRB-

clone

Novocastra

-RRB-

-LRB-

polyclonal

dilution

Cell

Signalling

Technology

Beverly

MA

USA

-RRB-

ubiquitine

-LRB-

polyclonal

-RRB-

α-synuclein

-LRB-

polyclonal

Affiniti/SA

-RRB-

-LSB-

polyclonal

rabbit

dilution

Santa

Cruz

Biotechnology

-LRB-

SCBT

-RRB-

Santa

Cruz

CA

USA

-RSB-

phosphorylated

-LRB-

p

-RRB-

PERK

-LRB-

polyclonal

rabbit

dilution

SCBT

-RRB-

and

phosphorylated

-LRB-

p

-RRB-

-LRB-

polyclonal

rabbit

dilution

Sigma

-RRB-

Paraffin

sections

were

routinely

stained

with

HE

LFB

and

Nissl

Cerebellar

sections

were

silver

stained

according

to

Bielschowsky

Immunocytochemistry

was

carried

out

on

paraffin-embedded

tissue

as

previously

described

-LSB-

-RSB-

The

sections

were

incubated

for

h

at

room

temperature

followed

by

incubation

at

°C

overnight

with

primary

antibodies

Single-label

immunocytochemistry

was

performed

using

avidin-biotin

peroxidase

method

Chromogen

or

-LRB-

AEC

Zymed

San

Francisco

CA

-RRB-

was

used

and

nuclei

were

stained

with

hematoxylin

Sections

incubated

without

the

primary

Ab

with

preimmune

sera

were

essentially

blank

For

double

labeling

-LRB-

with

ubiquitin

and

HLA-DR

-RRB-

sections

were

incubated

for

h

at

RT

with

Alexa

Fluor

and

Alexa

Fluor

-LRB-

anti-rabbit

IgG

anti-mouse

IgG

Molecular

probes

Eugene

USA

-RRB-

The

sections

were

then

analyzed

by

means

of

a

laser

scanning

confocal

microscope

-LRB-

Bio-Rad

Hercules

CA

USA

-RRB-

equipped

with

an

argon-ion

laser

Results

Details

of

each

patient

are

given

in

Table

Figure

shows

the

pedigree

which

links

patients

and

to

a

common

ancestry

Parents

of

patients

and

are

consanguineous

The

three

parental

couples

originate

from

the

Dutch

community

in

Volendam

which

has

a

high

degree

of

consanguinity

They

link

genetically

to

one

or

two

ancestral

couples

I

I

and

III

III

born

in

the

eighteenth

century

A

genealogical

link

between

these

ancestral

couples

who

carry

identical

family

names

is

suggested

but

yet

unproven

Fig.

linking

patients

and

in

the

present

report

IX

and

IX

were

reported

before

-LSB-

-RSB-

Cerebellar

cortex

The

cerebellar

hemispheres

are

severely

affected

in

all

On

macroscopic

examination

all

major

lobes

are

reduced

in

width

and

dorsoventrally

flattened

Size

was

best

preserved

in

the

transverse

plane

This

asymmetric

diminution

in

size

and

the

relative

sparing

of

the

vermis

lend

the

cerebella

a

butterfly

or

batwing

aspect

-LRB-

Fig.

-RRB-

Four

of

the

six

cerebella

-LRB-

cases

-RRB-

are

sectioned

perpendicular

to

the

folia

with

the

line

of

the

sectioning

running

from

the

culmen

to

the

posterior

pole

These

sections

include

the

flocculus

In

two

cases

-LRB-

-RRB-

transverse

sections

of

hemispheres

and

vermis

are

prepared

Low

magnification

-LRB-

Fig.

-RRB-

shows

the

diminished

size

of

the

cerebellar

hemispheres

folial

shortening

and

diminished

number

of

folial

branches

compared

to

a

control

-LRB-

Fig.

b

-RRB-

In

the

most

affected

case

-LRB-

Fig.

h

-RRB-

no

branches

are

seen

at

all

while

in

a

less

affected

case

folial

branches

are

reduced

to

two

or

less

-LRB-

Fig.

f

-RRB-

The

other

cerebella

displayed

regular

folial

branching

-LRB-

Fig.

d

-RRB-

though

less

in

quantity

than

the

control

specimen

-LRB-

Fig.

b

-RRB-

Fig.

folia

Stains

synaptophysin

Sagittal

sections

of

cerebellar

hemispheres

are

seen

on

the

left

and

detailed

views

on

the

right

Stunted

folial

growth

with

decreased

number

of

folial

branches

a

b

Male

years

with

accidental

death

and

normal

findings

on

autopsy

-LRB-

control

-RRB-

c

d

case

e

f

case

g

h

case

Marker

bars

on

the

left

cm

and

on

the

right

mm

The

vermis

is

fully

developed

in

all

cases

with

the

individual

lobules

well

developed

in

size

and

branching

pattern

Sagittal

sections

of

the

vermis

available

in

four

show

some

inequality

in

thickness

of

the

white

matter

cores

of

the

lobules

with

the

declive

folium

tuber

complex

the

best

preserved

part

in

three

-LRB-

cases

-RRB-

and

the

pyramis

in

one

-LRB-

case

Fig.

-RRB-

Fig.

cerebelli

case

Stains

a

HE

b

GFAP

c

neurofilament

protein

d

vimentin

a

c

Adjacent

sections

with

circumscript

segments

of

loss

of

all

layers

and

glial

reaction

Arrows

in

-LRB-

a

-RRB-

point

to

circumscript

areas

of

lost

cortex

d

Sharply

demarcated

foci

and

replacing

bands

of

astrocytes

possibly

modified

Bergmann

cells

Marker

bar

in

-LRB-

d

-RRB-

μm

On

microscopy

regressive

changes

are

present

in

all

with

the

vermis

relatively

spared

In

the

vermis

diffuse

but

moderate

loss

of

Purkinje

cells

internal

granule

and

folial

white

matter

is

seen

in

all

lobules

Two

cases

present

characteristic

sharply

delimited

foci

with

full

thickness

loss

of

vermal

cerebellar

cortex

with

astroglial

replacement

-LRB-

Fig.

-RRB-

The

hemispheric

cerebellar

cortex

is

affected

in

all

cases

by

variable

loss

of

Purkinje

cells

thinning

of

the

granule

cell

layer

and

inhomogeneous

loss

of

myelinated

fibers

within

folia

and

central

white

matter

The

remaining

axons

are

well

myelinated

and

stain

for

SMI

and

MBP

In

addition

to

diffuse

cortical

changes

sharply

delimited

areas

of

up

to

mm

in

width

with

loss

of

all

cortical

neurons

are

found

in

cases

-LRB-

-RRB-

These

areas

locate

mostly

but

not

exclusively

to

stretches

of

cortex

connecting

adjacent

folia

The

number

of

these

areas

varies

between

two

per

whole

cerebellar

hemispheric

section

-LRB-

-RRB-

to

ten

or

more

-LRB-

Fig.

-RRB-

In

one

case

-LRB-

-RRB-

representing

the

oldest

surviving

patient

destruction

of

the

whole

cerebellum

is

almost

complete

with

subtotal

loss

of

all

neuronal

elements

-LRB-

Fig.

-RRB-

Even

in

this

case

patches

of

whole

thickness

loss

of

cortex

alternate

with

segments

of

cortex

where

gliosis

still

preserves

the

original

frame

of

cortical

structure

-LRB-

Fig.

-RRB-

In

all

cases

the

flocculus

belongs

to

the

relatively

spared

areas

of

the

hemispheric

cortex

In

one

case

the

sparing

of

the

flocculus

stands

out

in

sharp

contrast

to

the

rest

of

the

hemispheric

cortex

-LRB-

Fig.

-RRB-

Fig.

hemispheric

cortex

with

segmental

lesions

Stains

a

h

synaptophysin

b

d

vimentin

e

g

i

GFAP

f

Case

opposing

arrows

mark

lesions

with

loss

of

all

cortical

layers

-LRB-

a

-RRB-

and

glial

replacement

-LRB-

b

-RRB-

c

d

Case

segment

of

whole

thickness

loss

of

cortex

is

replaced

by

a

single

layer

of

evenly

spaced

astrocytes

their

topography

compatible

with

modified

Bergmann

cells

e

f

g

Case

segmental

loss

of

cortex

with

glial

replacement

marked

by

opposing

arrows

in

-LRB-

f

-RRB-

higher

magnification

shows

a

group

of

remaining

isolated

granule

cells

h

i

Case

segmental

loss

of

cortex

on

top

of

a

folium

in

moderately

hypoplastic

cortex

Marker

bars

a

c

e

g

i

mm

d

mm

f

μmFig

Cerebellar

hemispheric

cortex

case

synaptophysin

Floccular

cortex

shows

broader

folial

cores

and

denser

granule

cell

population

than

other

parts

of

the

cortex

Dentate

nucleus

The

dentate

nucleus

is

affected

in

all

cases

by

major

loss

of

dentate

neurons

and

by

residual

cells

being

grouped

in

islands

-LRB-

Figs.

-RRB-

Islands

as

well

as

individual

neurons

are

surrounded

by

whorls

of

nerve

fibers

Residual

neurons

and

the

proximal

parts

of

their

dendrites

stain

positive

for

and

synaptophysin

Synaptophysin

staining

is

abundant

on

neurons

and

proximal

dendrites

signifying

retained

network

activity

in

surviving

neurons

A

peculiar

vacuolization

surrounds

individual

dentate

neurons

-LRB-

Fig.

b

-RRB-

A

proportion

of

these

vacuoles

stain

positive

for

calbindin

suggestive

of

swollen

axon

terminals

-LRB-

Fig.

d

-RRB-

Cerebellar

white

matter

surrounding

the

dentate

nucleus

on

the

inside

and

outside

shows

thickened

axons

with

diameters

up

to

μm

-LRB-

Fig.

-RRB-

The

other

internal

cerebellar

nuclei

could

not

be

identified

with

certainty

Fig.

nucleus

case

Loss

of

normal

outline

with

remaining

dentate

neurons

clustered

in

islands

Marker

bar

mmFig

nucleus

details

Stains

a

b

MBP

c

d

calbindin

e

synaptophysin

f

a

Case

fine

vacuolation

surrounding

neurons

b

Case

myelinated

fibers

separate

individual

neurons

and

their

surrounding

vacuoles

c

d

Cases

some

vacuoles

are

decorated

by

antibody

e

Case

concentration

of

vesicles

-LRB-

dots

-RRB-

indicates

high

synaptic

density

on

soma

and

dendrites

of

surviving

neuron

f

Case

thickened

axons

in

hilus

of

dentate

nucleus

Marker

bars

μm

Cystic

degeneration

of

the

cerebellum

In

two

cases

destruction

resulted

in

cyst

formation

-LRB-

cases

-RRB-

MR

investigation

shows

cystic

change

in

the

cerebellar

hemispheres

of

patient

at

the

age

of

month

-LRB-

Fig.

-RRB-

On

autopsy

the

cysts

are

collapsed

and

can

only

be

retrieved

by

serial

sectioning

The

border

of

the

cyst

is

lined

by

reactive

astrocytes

and

macrophages

-LRB-

Fig.

c

-RRB-

In

case

cyst

formation

compounds

the

subtotal

loss

of

cortex

dentate

nuclei

and

fiber

tracts

A

remarkable

involvement

of

small

sized

vessels

not

present

in

the

other

cases

consists

of

intimal

proliferation

and

splitting

of

the

elastica

interna

This

abnormality

is

only

present

in

the

cerebellum

and

its

surrounding

arachnoid

vessels

-LRB-

Fig.

-RRB-

Electron

microscopy

fails

to

show

storage

material

or

enlarged

lysosomes

-LRB-

not

shown

-RRB-

Double

labeling

for

ubiquitin

and

HLA-DR

shows

that

ubiquitin

is

mainly

present

in

the

vessel

walls

and

only

in

minor

part

associated

with

macrophages

-LRB-

HLA-DR

Fig.

-RRB-

Vascular

abnormalities

are

absent

in

other

parts

of

the

brain

and

in

internal

organs

including

the

kidneys

Fig.

cyst

case

a

Vimentin

c

HLA-DR

a

Collapsed

cyst

seamed

by

astroglial

reaction

b

axial

image

of

posterior

fossa

of

patient

at

month

age

shows

the

cysts

in

expanded

state

c

Macrophage

reaction

on

the

inside

of

the

cystFig

changes

case

Stains

a

GFAP

b

HLA-DR

c

double

labeling

for

HLA-DR

-LRB-

green

-RRB-

and

ubiquitine

-LRB-

red

-RRB-

confocal

laser

microscopy

d

ubiquitin

a

Transverse

cerebellar

section

with

cortical

and

subcortical

gliosis

loss

of

the

subcortical

and

central

cerebellar

structure

in

part

cystic

broken

cortical

lining

in

places

suggest

segmental

lesions

similar

to

other

cases

before

ultimate

decay

b

abundance

of

reactive

macrophages

predominantly

in

and

surrounding

blood

vessels

with

thickened

vessel

walls

c

ubiquitin

is

mostly

restricted

to

vessel

wall

and

macrophages

are

partly

positive

for

ubiquitin

d

neuron

of

nucleus

magnus

raphes

-LRB-

pontine

tegmentum

-RRB-

with

cytoplasmic

inclusion

and

positivity

extending

into

the

neurite

Marker

bars

b

μm

c

μm

Inferior

olivary

nuclei

The

inferior

olivary

nucleus

and

the

dorsal

and

medial

accessory

olivary

nuclei

are

identified

in

each

case

Mature

folding

of

the

inferior

olivary

nucleus

is

present

in

the

lateral

leaf

and

reduced

in

the

medial

leaf

in

one

case

-LRB-

Fig.

-RRB-

In

all

others

folding

is

reduced

in

both

leaves

the

medial

leaf

being

more

affected

than

the

lateral

Discontinuous

loss

of

parts

of

the

nerve

cell

band

is

noted

in

each

case

best

shown

by

staining

-LRB-

Fig.

-RRB-

In

one

case

-LRB-

-RRB-

nearly

all

olivary

neurons

including

the

accessory

olivary

nuclei

are

lost

Part

of

this

loss

is

mirrored

by

vimentin

positive

astrocytes

forming

a

ghost

image

of

the

lost

neuronal

framework

-LRB-

Fig.

d

-RRB-

Macrophages

are

present

within

and

around

the

vanished

structure

but

not

in

a

topographically

distinct

pattern

In

all

cases

internal

and

external

olivary

fibers

are

reduced

or

lost

entirely

Fig.

olivary

nucleus

Stains

a

c

d

vimentin

case

and

segmental

loss

of

inferior

olivary

nucleus

with

intact

dorsal

-LRB-

a

-RRB-

and

medial

-LRB-

b

-RRB-

accessory

nuclei

c

Case

Complete

loss

of

nucleus

including

accessory

nuclei

only

sparse

isolated

neurons

remaining

indicated

by

arrowheads

inset

shows

magnified

solitary

olivary

neuron

d

Case

Outline

of

lost

lateral

leaf

reveals

mature

folding

Marker

bars

mm

Pons

Ventral

pons

Similar

findings

are

present

in

all

cases

Diminution

in

size

of

the

pons

to

about

half

or

less

of

its

size

in

transverse

diameter

is

entirely

due

to

loss

of

its

ventral

compartment

The

majority

of

ventral

pontine

neurons

and

transverse

pontine

fibers

are

lost

with

sparing

of

the

long

motor

and

sensory

tracts

Prominent

gliosis

-LRB-

Vim

and

GFA

-RRB-

and

microglial

activation

are

found

in

the

affected

region

Staining

for

synaptophysin

and

calbindin

in

the

remaining

neurons

does

not

reveal

specific

patterns

Caspase

and

ubiquitin

staining

is

negative

in

the

few

remaining

neurons

Pontine

tegmentum

No

abnormalities

are

encountered

in

the

cranial

nerve

nuclei

raphe

or

central

tegmental

tract

Ubiquitin

staining

in

the

reticular

nuclei

especially

the

nucleus

raphes

magnus

in

case

reveals

numerous

neurons

with

ubiquitin-positive

cytoplasmic

inclusions

staining

is

negative

in

all

cases

Other

findings

in

the

brainstem

Total

absence

of

the

medullary

arcuate

nuclei

is

found

in

all

cases

Widespread

presence

of

lipopigment

-LRB-

lipofuscin

-RRB-

is

observed

in

all

cases

especially

in

larger

neurons

-LRB-

dentate

nuclei

olivary

nuclei

pontine

neurons

brainstem

tegmentum

and

cerebral

cortex

-RRB-

even

in

the

youngest

individuals

Staining

for

α-synuclein

was

negative

in

the

brainstem

as

well

as

in

the

cerebellum

in

all

cases

Midbrain

thalamus

and

basal

ganglia

Nucleus

ruber

has

diminished

size

and

cell

density

in

case

This

case

contrasts

with

other

cases

by

its

subtotal

destruction

of

the

cerebellum

In

all

available

cases

-LRB-

-RRB-

the

caudate

nuclei

are

macroscopically

atrophic

There

is

a

general

increase

of

astrocytes

and

microglia

throughout

the

basal

ganglia

and

thalami

Myelination

is

normal

No

specific

abnormalities

are

found

In

one

case

severe

necrotic

changes

are

found

in

the

globus

pallidus

representing

acute

postischemic

damage

Cerebral

cortex

Macroscopic

gyral

atrophy

is

seen

in

three

of

five

cortices

available

for

study

The

temporal

lobes

are

affected

in

one

-LRB-

case

-RRB-

the

frontal

and

temporal

lobes

in

one

-LRB-

case

-RRB-

and

all

lobes

in

one

-LRB-

case

-RRB-

Myelination

is

normal

in

large

sections

-LRB-

luxol

fast

blue

-RRB-

even

in

atrophic

parts

Cortical

development

appears

normal

on

routine

staining

A

diffuse

increase

of

reactive

astroglia

and

activated

microglia

is

seen

throughout

the

neocortex

and

subcortical

and

central

white

matter

in

all

cases

Except

for

postischemic

changes

in

one

case

-LRB-

-RRB-

the

hippocampal

and

dentate

gyri

are

normal

Olivopontocerebellar

hypoplasia

Case

this

male

baby

was

born

at

weeks

ga.

The

amount

of

amniotic

fluid

was

normal

Spontaneous

respiration

was

absent

He

had

trismus

severe

tremor/clonus

and

varus

deformity

of

the

legs

On

autopsy

the

supratentorial

parts

of

the

brain

are

small

size

but

of

normal

appearance

while

the

cerebellum

and

brainstem

are

disproportionately

small

The

cerebellum

is

remarkable

for

its

complete

sparing

of

the

nodulus

and

flocculus

its

severe

cortical

depletion

on

the

dorsal

side

and

hypoplastic

cortical

development

on

the

ventral

side

of

the

hemispheres

-LRB-

Fig.

-RRB-

Folial

development

of

the

neocerebellum

is

severely

restricted

with

few

folia

on

its

dorsal

parts

Microscopic

examination

of

the

flocculus

and

vermis

is

normal

Neocerebellar

hemispheric

cortex

shows

isomorphic

gliosis

through

transformed

Bergmann

cells

-LRB-

Fig.

-RRB-

staining

shows

some

retained

neuronal

elements

-LRB-

Fig.

d

-RRB-

The

dentate

nuclei

are

broken

up

into

small

islands

that

can

only

be

retrieved

by

neuron-specific

staining

This

reveals

a

few

small

clusters

with

well-developed

dentate

neurons

left

in

place

-LRB-

Fig.

f

-RRB-

The

inferior

olivary

nuclei

have

the

form

of

horseshoes

without

folding

and

with

the

segments

of

the

nucleus

missing

on

both

sides

-LRB-

Fig.

-RRB-

The

accessory

olivary

nuclei

are

spared

The

ventral

pons

is

severely

underdeveloped

with

most

of

the

pontine

nuclei

missing

dense

gliosis

and

absence

of

staining

A

diffuse

astrocytic

gliosis

is

found

throughout

the

cerebral

hemispheres

especially

affecting

the

subcortical

white

matter

and

the

fiber

tracts

of

the

striatum

The

neocortex

is

unremarkable

Fig.

case

Stains

a

e

synaptophysin

b

vimentin

c

d

f

a

Whole

mount

transverse

section

of

cerebellum

and

medulla

oblongata

denuded

hemispheric

cerebellar

cortex

and

striking

preservation

of

nodulus

and

flocculi

b

Hemispheric

cortex

lined

by

a

single

row

of

fibrous

astrocytes

c

Empty

cerebellar

cortex

arrows

indicate

some

remaining

neurons

d

Higher

magnification

of

c

arrowhead

indicates

bipolar

neuron

possibly

basket

cell

arrow

indicates

malpositioned

Purkinje

cell

e

Overview

of

cerebellar

hemisphere

arrows

at

residues

of

dentate

nucleus

f

Single

residual

island

of

dentate

nucleus

with

well-developed

neurons

Marker

bars

b

mμ

c

mm

d

mμ

e

mm

f

mμFig

case

Stain

synaptophysin

Medulla

oblongata

with

olivary

nuclei

Inferior

olivary

nucleus

with

complete

lack

of

folding

Lateral

and

medial

leaves

with

segmental

defects

Medial

accessory

olive

is

preserved

in

its

dorsal

part

and

shows

discontinuities

in

its

ventral

part

Marker

bar

mm

Unfolded

protein

response

We

previously

observed

morphological

changes

in

the

endoplasmic

reticulum

-LRB-

ER

-RRB-

in

a

biopsy

of

a

patient

by

electron

microscopy

This

led

us

to

investigate

whether

the

unfolded

protein

response

-LRB-

UPR

-RRB-

a

stress

response

of

the

ER

-LSB-

-RSB-

is

activated

in

neurons

of

patients

The

UPR

was

studied

in

sections

from

the

frontal

cortex

-LRB-

patients

-RRB-

and

the

cerebellar

hemispheres

which

included

the

dentate

nucleus

-LRB-

patients

-RRB-

As

a

marker

of

induction

of

the

UPR

we

used

an

antibody

specific

for

the

phosphorylated

pancreatic

ER

kinase

-LRB-

pPERK

-RRB-

which

is

an

ER

stress

transducer

that

is

only

phosphorylated

during

the

UPR

Previously

we

have

used

this

method

to

show

UPR

activation

in

the

brain

of

patients

with

Alzheimer

's

disease

-LSB-

-RSB-

or

Parkinson

's

disease

-LSB-

-RSB-

We

find

no

immunopositive

staining

for

pPERK

in

any

of

the

patients

neither

in

the

cortex

nor

in

the

cerebellum

Eukaryotic

initiation

factor

2α

-LRB-

-RRB-

is

the

substrate

of

pPERK

and

using

a

antibody

increased

staining

intensity

is

observed

as

compared

to

the

control

patient

in

the

Purkinje

cells

of

patients

and

This

is

not

found

in

patient

and

severe

degeneration

in

patients

and

precludes

proper

analysis

Apart

from

the

fact

that

differences

in

intensities

between

pathological

samples

are

not

always

readily

interpretable

this

does

not

provide

evidence

for

UPR

activation

in

the

absence

of

pPERK

reactivity

because

other

kinases

can

also

phosphorylate

The

ER

chaperone

BiP

is

upregulated

during

the

UPR

but

in

accordance

with

the

pPERK

data

no

obvious

differences

in

levels

are

observed

In

summary

we

find

no

evidence

for

activation

of

the

UPR

Discussion

Cerebellar

cortex

dentate

nucleus

and

olivary

complex

The

series

of

six

cases

presents

developmental

and

regressive

abnormalities

of

the

cerebellar

cortex

dentate

nuclei

and

olivary

nuclei

Cerebellar

cortical

findings

range

between

short

unbranched

folia

in

the

least-developed

cerebellum

-LRB-

case

-RRB-

to

shortened

folia

with

a

decreased

number

of

branches

In

the

most

affected

cerebella

poorly

developed

folia

alternate

with

flat

stretches

of

surface

that

lack

proper

cortical

lining

A

similar

observation

is

reported

in

cases

-LSB-

-RSB-

OPCH

-LSB-

-RSB-

a

case

classified

as

neocerebellar

hypoplasia

with

combined

olivo-ponto-dentatal

degeneration

-LSB-

-RSB-

and

with

spasticity

-LSB-

-RSB-

Reactive

glial

changes

were

reported

in

-LSB-

-RSB-

All

empty

segments

of

cortex

in

the

present

study

have

a

similar

appearance

a

single

evenly

spaced

row

of

transformed

Bergmann

cells

positively

staining

for

vimentin

Synaptophysin

and

staining

reveal

the

sparse

presence

of

nerve

fibers

This

is

the

first

time

this

patten

is

observed

by

immunohistochemistry

The

earliest

stages

of

cerebellar

development

are

probably

normal

because

they

require

the

coordinate

development

and

juxtaposition

of

two

cell

types

the

Purkinje

cells

that

arise

by

radial

outward

migration

and

the

external

granule

cells

that

arise

by

tangential

migration

and

enter

the

future

cortex

to

become

internal

granule

cells

-LSB-

-RSB-

The

microscopic

structure

of

the

empty

segments

betrays

previous

cortical

development

The

earliest

estimate

for

the

loss

of

cortex

in

these

segments

follows

from

the

afolial

stretches

in

patients

and

that

represent

a

stage

of

cerebellar

development

prior

to

or

coinciding

with

beginning

folial

outgrowth

around

weeks

gestational

age

in

the

human

fetus

-LSB-

-RSB-

Cortical

segmental

loss

however

is

not

restricted

to

afolial

segments

but

is

also

seen

in

more

developed

hemispheric

cortex

-LRB-

e.g.

Fig.

i

-RRB-

and

mature

vermis

This

suggests

a

time

frame

for

segmental

loss

of

cerebellar

cortex

from

incipient

foliation

to

encompass

late

cortical

development

It

can

not

be

established

presently

whether

segmental

loss

is

a

completed

process

or

still

active

at

the

time

of

demise

No

microscopic

vascular

changes

are

seen

in

the

vicinity

of

segmental

lesions

and

their

distribution

is

not

restricted

to

arterial

boundary

zones

Also

cerebellar

vascular

changes

observed

in

one

case

-LRB-

-RRB-

are

not

found

in

any

of

the

other

cases

and

therefore

offer

no

explanation

for

segmental

cortical

lesions

The

spectrum

of

changes

in

the

inferior

olivary

nucleus

in

this

series

similarly

encompasses

early

and

late

developments

In

five

out

of

six

cases

the

degree

of

folding

is

immature

while

in

the

sixth

case

-LRB-

case

-RRB-

a

mature

folding

pattern

of

the

nucleus

can

be

gauged

by

observing

the

outline

of

the

astroglial

scar

According

to

one

study

in

human

fetal

material

-LSB-

-RSB-

the

mature

form

of

the

nucleus

is

reached

with

a

mm

crown

heel

length

i.e.

weeks

Segmental

loss

is

observed

in

cases

in

the

olivary

nucleus

and

the

sequence

of

progressive

loss

may

have

been

similar

in

the

remaining

oldest

case

-LRB-

case

-RRB-

with

loss

of

the

entire

nucleus

Segmental

lesions

in

the

inferior

olivary

nucleus

in

PCH

were

reported

in

-LSB-

-RSB-

The

consistency

of

this

finding

is

shown

in

the

present

series

using

immunohistochemistry

The

third

structure

with

a

segmental

pattern

of

loss

is

the

dentate

nucleus

We

have

no

evidence

in

this

series

of

a

normal

outline

of

the

dentate

nucleus

during

any

phase

of

its

development

One

previous

report

-LSB-

-RSB-

however

relates

a

glial

scar

linking

the

islands

of

the

dentate

in

PCH

We

examined

a

case

classified

as

OPCH

and

on

comparison

to

the

series

found

important

similarities

-LRB-

-RRB-

flat

stretches

of

cerebellar

cortex

and

ghosts

of

primitive

folia

carrying

microscopic

remnants

of

a

pre-existent

developed

cortex

-LRB-

-RRB-

dentate

islands

with

well-developed

neurons

-LRB-

-RRB-

segmental

loss

in

the

inferior

olivary

nucleus

-LRB-

-RRB-

sparing

of

nodulus

and

flocculus

These

findings

reveal

a

remarkable

analogy

to

the

segmental

cortical

lesions

in

Differences

between

and

OPCH

are

-LRB-

-RRB-

the

horseshoe

appearance

of

the

inferior

olivary

nucleus

compared

to

the

diminished

folding

in

indicates

earlier

onset

of

the

pathological

process

in

OPCH

-LRB-

-RRB-

the

extent

of

afolial

cerebellar

cortex

Regressive

changes

In

addition

to

developmental

abnormalities

some

findings

indicate

an

ongoing

process

such

as

cytoplasmic

ubiquitin

positivity

in

pontine

tegmental

neurons

in

case

the

widespread

presence

of

astrocytic

and

microglial

reaction

and

the

abundance

of

lipopigment

e.g.

in

cerebral

cortical

cerebellar

cortical

dentate

and

olivary

neurons

Cyst

formation

in

the

cerebellar

white

matter

-LRB-

cases

-RRB-

and

vascular

changes

in

the

cerebellum

-LRB-

case

-RRB-

also

signify

a

regressive

element

in

A

previously

made

observation

on

darkened

stretches

of

endoplasmic

reticulum

in

cortical

neurons

in

a

cortical

biopsy

-LSB-

-RSB-

prompted

us

to

investigate

the

ER

stress

response

with

negative

results

Comparison

of

to

other

neurodegenerative

disorders

The

nearest

equivalent

in

neuropathological

terms

to

is

the

group

previously

named

olivo-ponto-cerebellar

atrophy

-LRB-

OPCA

-RRB-

presently

known

as

spinocerebellar

ataxia

-LRB-

SCA

-RRB-

While

olivary

and/or

dentate

nucleus

may

be

affected

of

the

typical

features

especially

dentate

fragmentation

segmental

lesions

of

the

cerebellar

cortex

and

olivary

nucleus

is

part

of

the

pathological

spectrum

of

these

disorders

-LSB-

-RSB-

Pathological

studies

on

autosomal

recessive

or

X-linked

central

nervous

system

disorders

with

onset

in

childhood

and

predominant

impact

on

the

cerebellum

include

the

Høyeraal-Hreidarsson

syndrome

-LSB-

-RSB-

progressive

encephalopathy

hypsarrhythmia

optic

atrophy

-LRB-

PEHO

-RRB-

-LSB-

-RSB-

primary

granular

cell

degeneration

of

the

cerebellum

-LSB-

-RSB-

and

infantile

onset

spinocerebellar

ataxia

with

neuropathy

-LRB-

IOSCA

-RRB-

-LSB-

-RSB-

These

disorders

display

cerebellar

atrophy

rather

than

hypoplasia

lack

the

segmental

cortical

anomalies

the

characteristic

fragmentation

of

the

dentate

and

the

segmental

loss

of

the

olivary

nucleus

described

above

with

the

possible

exception

of

IOSCA

in

which

segmental

loss

of

the

olivary

nucleus

is

documented

-LSB-

-RSB-

Neurometabolic

disorders

of

glycosylation

especially

congenital

disorder

of

glycosylation

-LRB-

CDG

-RRB-

type

and

the

alpha-dystroglycanopathies

manifest

cerebellar

hypoplasia

often

in

combination

with

pontine

hypoplasia

Neuropathological

findings

in

do

not

include

circumscript

dentate

olivary

or

cerebellar

cortical

defects

-LSB-

-RSB-

Alpha-dystroglycanopathies

combine

a

severe

type

of

congenital

muscular

dystrophy

with

cerebral

cerebellar

and

brainstem

malformation

and

deficiency

of

alpha-dystroglycan

-LRB-

alpha

DG

-RRB-

-LSB-

-RSB-

Alpha

DG

deficiency

in

this

group

probably

causes

a

tangential

neuronal

migration

defect

affecting

migration

from

the

rhombic

lip

into

the

cerebellum

and

ventral

pons

The

resulting

pathology

-LSB-

-RSB-

causes

heterotopia

and

impaired

foliation

in

the

cerebellum

Summarizing

previous

and

present

data

on

this

disorder

bears

a

number

of

features

not

encountered

in

other

disorders

with

the

exception

of

which

appears

to

be

closely

related

but

for

the

involvement

of

spinal

anterior

horn

cells

in

the

latter

Neural

mechanisms

A

question

may

be

raised

whether

segmental

lesions

in

the

three

nuclear

systems

as

observed

in

could

be

effected

through

a

single

mechanism

Cerebellar

cortex

olivary

nuclei

and

dentate

nuclei

are

functionally

linked

through

the

climbing

fiber

system

which

is

topographically

organized

in

sagittal

modules

each

formed

by

a

segment

of

the

olivary

nuclear

complex

its

collaterals

to

the

cerebellar

nuclei

including

the

dentate

nucleus

and

a

strip

of

cerebellar

cortex

-LSB-

-RSB-

A

sagittal

parcelleation

of

the

cerebellum

in

biochemically

distinct

regions

however

develops

independent

of

the

olivo-cerebellar

projection

-LSB-

-RSB-

Lesions

of

the

olivary

complex

are

not

known

to

cause

segmental

lesions

in

other

parts

of

the

climbing

fiber

system

Experimental

administration

of

the

drugs

harmaline

or

ibogaine

to

rats

results

in

tremor

and

loss

of

sagittal

strips

of

vermis

The

role

of

the

olivary

nucleus

in

this

type

of

segmental

damage

is

highlighted

by

prior

chemical

ablation

of

the

inferior

olivary

nucleus

by

pyridine

which

prevents

the

segmental

damage

from

occurring

Overexcitation

of

the

olivary

nucleus

rather

than

primary

loss

is

apparently

required

for

this

type

of

lesion

to

occur

-LSB-

-RSB-

The

type

of

lesion

reported

in

rodents

is

reminescent

of

the

segmental

lesions

in

One

argument

in

support

of

a

possible

role

of

the

olivo-cerebellar

projection

in

is

the

selectivity

of

the

cerebellar

cortex

involved

in

which

the

vermis

and

flocculus

are

relatively

spared

The

climbing

fiber

output

of

the

inferior

olivary

nucleus

to

the

cerebellum

is

mainly

directed

to

the

cerebellar

hemispheres

The

flocculus

and

nodulus

both

relatively

spared

in

and

in

OPCH

are

reciprocally

connected

to

the

vestibular

nuclei

-LSB-

-RSB-

We

have

not

found

abnormalities

in

the

vestibular

nuclei

in

our

series

In

addition

to

cortical

segmental

loss

a

more

diffuse

kind

of

degeneration

is

found

which

includes

loss

of

cortical

elements

such

as

Purkinje

cells

and

granule

cells

This

lends

a

moth-eaten

aspect

to

the

myelinated

nerve

bundles

in

the

cores

of

folia

and

central

cerebellar

white

matter

similar

to

the

more

common

cerebellar

degenerations

such

as

in

spino-cerebellar

ataxias

Some

findings

such

as

gliosis

in

the

central

parts

of

the

cerebellum

and

axonal

swelling

suggest

an

ongoing

process

Study

of

the

cell

somata

of

the

nucleus

dentatus

reveals

some

peculiarities

in

all

patients

where

the

nucleus

could

be

identified

not

seen

in

controls

such

as

perisomatic

vacuolation

that

was

even

seen

on

routine

HE

staining

The

presence

of

calbindin

in

some

of

these

vacuoles

suggests

swollen

nerve

endings

Genetics

and

epigenetics

Varying

degrees

of

hypoplastic

folial

development

are

found

in

three

patients

-LRB-

-RRB-

who

share

the

same

ancestors

-LRB-

Fig.

h

-RRB-

This

indicates

that

modifying

factors

bear

on

the

expression

of

this

genetic

condition

Patient

the

patient

with

the

longest

survival

who

died

at

years

differed

from

other

patients

in

having

vascular

anomalies

in

the

cerebellum

This

phenomenon

may

be

caused

by

an

unknown

age-bound

complication

of

the

disease

However

a

genotype

distinct

from

the

others

can

not

be

excluded

pending

the

unraveling

of

the

genetic

basis

of

B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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cerebellum
pontocerebellar hypoplasia
olivary nucleus
pons