Sorachai Srisuma MD PhD Sorachai Srisuma MD PhD ssrisuma@rics.bwh.harvard.edu

Sorachai Srisuma MD PhD
Sorachai Srisuma, MD, PhD
ssrisuma@rics.bwh.harvard.edu

Both are performed under flexible
bronchoscopy.
Histological analyses of airway tissue provide
◦ The information of pathological changes.
◦ Assess potential therapeutic effects in pharmacological
studies.
EBB allow the assessment of large airway, but
not the entire thickness.
TBB allow the assessment of the distal lung
including the distal airway wall and the alveolar
tissues.
Limitations of TBB are major complications
such as bleeding and pneumothorax.

Epithelial Basement Subepithelial
alteration membrane fibrosis
thickness
Asthma Detachment +++ +++
COPD metaplasia + ++
+ represents the degree of association with disease

Mucus gland Smooth Angiogenesis
hyperplasia muscle
mass
Asthma ++ +++ +++
COPD +++ ++ +
+ represents the degree of association with disease

Resected tissue needs to be preserved
◦ Frozen
◦ Embedded in paraffin
Electron microscopy
Histochemical staining
Immunohistochemistry
In situ hybridization

H&E (Haematoxylin and eosin) staining
Total collagen stain
◦ Sirius red
◦ Van Gieson
◦ Masson-trichrome
PAS (periodic-acid shiff) staining is used to
visualize the mucus glands or mucuscontaining
cells.
Elastin staining
Hypoxia staining (pimonidazole
hydrochloride)

Bronchiole –H&E staining

PAS staining in mouse lung

Hypoxia of airway epithelial cells
Bronchiole –H&E staining
β-ENaC overexpressing
mice
wild type mice

It allows detection of specific proteins using
specific antibody.
The signal detected by IHC reflects the
tissue expression of proteins.

SERPINE2 (PN1) localization
in mouse lungs

Immunofluorescence

It allows detection of specific mRNA using
specific probes (oligonucleotides).
The signal detected by ISH reflects the
tissue and cellular l expression of mRNA.

Bronchoalveolar lavage (BAL)
Induced sputum
Exhaled breath condensate
Blood
Urine

Is performed under flexible bronchoscopy at
the same time as EBB.
Is routinely used to study cellular composition
and to measure the levels of
cytokines/chemokines in the distal airway and
alveoli.
Cannot differentiate the individual components
of the distal airway and alveolar compartments.
The cellular component mainly represents the
luminal inflammatory cells and some bronchial
epithelial cells.

Stained BAL cells

Stained BAL cells

Hypertonic saline-induced sputum
Is used to evaluate airway inflammation in
the central airway.
Can be used to study cellular components
from the mucus.
Soluble remodeling-associated proteins,
such as procollagen synthesis peptides,
MMP, TIMP and cytokines, can be detected
in the sputum supernatants.

Is a noninvasive method and reflects the
composition of the fluid lining the airway.
Several markers including hydrogen
peroxide, leukotrienes, prostaglandins,
isoprostanes, nitro oxide-derived products
and hydrogen ions have been measured
successfully in EBC.

Exhaled breath condensate
collecting system
stem

Indirect monitoring of lung inflammation
Paolo Montuschi
Nature Reviews Drug Discovery 1, 238-242 (March 2002)
a | Exhaled-breath condensate (EBC) collecting system, which consists of a
glass condensing chamber that contains a double wall of glass for which the
inner side of the glass is cooled by ice. EBC is collected between the two
glass surfaces and drops to the bottom of the outer glass container in a
liquid form. b | Schematic representation of a commercially available
condenser. Frozen EBC is collected in the collecting vial, as indicated by the
arrow.

Biomarkers of airway
inflammation

Protein quantification in fluids using specific
antibodies.
◦ Enzyme linked-immunosorbent assay (ELISA)
◦ Radio-immunoassayimmunoassay
◦ Western blot analysis
◦ Zymography

Primary cell isolation can be successfully
performed from human tissues.
◦ Epithelial cells
◦ Fibroblasts
◦ Smooth muscle cells
◦ Endothelial cells
To study the proliferative, contractile,
secretory, fibrogenic properties of cells
following a wide range of stimuli.

FEV 1 , FEC, FEV 1 /FVC
◦ Post-bronchodilator
◦ Challenge by bronchoconstrictor (Histamine,
cholinergic agonists)
◦ Related with airway basement membrane
thickness
Flow-volume loop analysis

Pathogenesis of asthma

Airway Hyperreactivity or
Airway hyperresponsiveness

Gas dilution (helium)

Plethysmograph is derived from the Greek
plethusmos (enlargement).
It is related closely to plethus (fullness) and
plethora (fullness).
It is used to determine
◦ Thoracic gas volume (TGV)
◦ Thoracic gas volume (TGV)
◦ Airway resistance (Raw)

The patient sits inside an airtight box of
known volume (Vbox).
inhales or exhales through a mouthpiece
connected to a shutter.
The pressure is monitored in 2 places
◦ Pressure in the box (Pbox)
◦ Pressure at the subject’s airway (Paw)

The subject makes respiratory efforts against
the closed shutter (pant), causing their chest
volume to expand and decompressing the air
in their lungs (∆Vlung).
The lung volume expanded (∆Vlung) must be
equal to the gas volume compressed in the
box, (∆Vbox)) at constant t temperature.
t
At the end of respiration, there is no flow, Paw
corresponds to alveolar pressure.

Flow signal
is negative
during inspiration
Box pressure signal
is positive
during inspiration

Flow signal
is positive
during expiration
Box pressure signal
is negative
during inspiration

The mouth shutter is closed.
Record mouth pressure vs box pressure for
determining TGV.
Mouth pressure
(cmH 2 0)
Box pressure (cmH 2 0)

At constant temperature,
P1V1 = P2V2
◦ P1 and V1 are initial pressure and volume.
◦ P2 and V2 are final pressure and volume.
PboxVbox = (Pbox+ ∆ Pbox) (Vbox- ∆ Vbox)
∆ Vbox can be calculated
In this case, ∆ Vbox equals ∆ Vlung
∆ Vlung can be measured directly by
connecting the mouthpiece to a spirometer,
instead of calculating it as ∆ Vbox.

P1V1 = P2V2
◦ P1 and V1 are initial pressure and volume.
◦ P2 and V2 are final pressure and volume.
Paw x FRC = (Paw-∆Paw) (FRC +∆ V Vlung) )
FRC can be calculated

Body plethysmography is particularly
appropriate for patients who have air spaces
within the lung that do not communicate
with the bronchial tree.
In these individuals, gas dilution methods of
measurement would give an erroneously low
volume reading.

The subject is asked to begin shallow
breathing at faster rate (1 breath per
second) and flow vs box pressure is
recorded.
d
Flow
(Liter/sec)
Box pressure (cmH 2 0)

When normal human expires fully to residual
volume, airways in the dependent lung regions tend
to close because of the high intrapleural pressure
there, when airways to upper lobes are still open.
The first gas entering the lungs on next inspiration
will go preferentially to upper lobes.
If the first gas inspired is a foreign gas, upper lobes
will have a much higher concentration of it than
lower lobes.
On the succeeding expiration, the lung volume at
which airways in lower lobes begin to close will be
marked by a sudden rise in concentration of the
foreign gas in expired air, since it is no longer
diluted by gas from lower lobes.

Ask the patient to breath out to residual volume.
Allow the patient to maximally breath in
100%O 2 to TLC.
Arrange the next inspiration so that the patient
first draws into his alveoli his dead space gas.
This dead space air (high N 2 concentration) goes
mainly to upper lobes.
O 2 then goes preferentially to lower lobes and
decreases the N2 concentration there.
N 2 concentration in the expired air can then be
measured continuously.

Single breath N 2 washout test
p.507-8, สรรวทยาเลม สรีรวิทยาเล่ม 2

An increase in the slope of the increase in
nitrogen percentage during the plateau
(alveolar-emptying) stage indicates
unevenness of alveolar l ventilation, and thus
abnormal VA/Q ratios.
Well-ventilated alveoli empty first.
Poorly-ventilated alveoli empty last.
Poorly-ventilated alveoli with high O 2 at base
of the lungs tends to lower the initial
plateau-nitrogen concentration.

When, as a result of dynamic compression, mid-size
airways in the base of the lungs collapse during the
"single breath nitrogen washout test," the
percentage of nitrogen in mixed exhaled air
markedly increases.
This is because once the basal airways close, only
the apical airways remain open and continue to
empty.
Because of their low compliance, the apical alveoli
receive less of the inhaled oxygen, thus when they
alone are emptying, the percentage of nitrogen in
the mixed exhaled air markedly increases.

The volume at which this occurs is called the closing
volume.
Closing volume is increased in obstructive lung
disease. It also increases with age and by 65 years-
of-age is nearly equal to the FRC. The increased
closing volume is a sign of "loss of interdependence"
due to loss of lung tissue.