scitp presentation27jul12 - toxpath.org concepts in target organ toxicologic pathology respiratory...

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Ninth Industrial Toxicology and Pathology Short Course

23‐27 July, 2012

Contemporary Concepts in Target Organ Toxicologic Pathology

Respiratory system

Respiratory Response to Toxic Injury (Lung)

Eric Wheeldon

Safety Assessment

GlaxoSmithKline

27 July 2012


Species Differences*Rat

Lung volume: 10.8mlELF volume : ~200µlNumber of alveoli: 19.7 x 106

Alveolar diameter: 70µM

DogLung volume: 1322mlELF volume: ~14.6mlNumber of alveoli: 1040 x 106

Alveolar diameter :126µM

HumanLung volume: 4341mlELF volume: ~24mlNumber of alveoli: 486 x 106

Alveolar diameter: 219µM

Structure* and Function*

architecture, branching

physiology

inhalation

air handling

gas exchange

Histology and response to injury*

cell types and distribution

vulnerability, clearance, inflammation

background changes

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Orders of branching

Length (mm) Volume (ml) Area (cm2)

1 220 80 2.0

14 130 71 13.9

3 5 43 76.1

3 3.6 865 281.0

3‐9 2.6 3000 7x105

Tidal Volume (ml) Minute Ventilation (ml/min)

Rat 2 150

Dog 200 3100

Monkey 20 750

Human 500 7000




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Bronchial Cell typesAirway Type

Lining Cell Secretory Cell

Bronchi Ciliated cell Goblet cell

Terminal bronchiole

Ciliated cell Clara cell

Alveolus Type I Type II


Epithelia of conducting airwaysSpecies Differences

Airway Species Cell types (approx %)$

Basal Ciliated Clara Serous Mucous

Trachea Rat 20 30 0 40 <1

Mouse 10 40 50 0 <1

Bronchi Rat 20 40 0 20 <1

Mouse 2 40 55 0 <1

Bronchiole Rat 0 60 40 0 0

Mouse 0 40 60 0 0

*other cells e.g., neurosecretory, eccrine also present

$ exact figures vary between sources


Mucociliary clearance

• Defense against insoluble particulates

• Ciliated cells ‐ co‐ordinated movement

• Secreting cells ‐mucus, serous


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• 2 layers of fluid

– sol ‐ lower level, watery allows cilia to move

– gel ‐ upper layer of mucus

• Lining fluid contains secretions ‐ IgA, lysozyme, anti‐proteases ‐ SLPI, elafin (elastase‐specific inhibitor), cytokines


http://reni.item.fraunhofer.de/reni/trimming/trimm.php


Particle impaction:

• Airflow changes direction• Particles near airway surface deposits by inertial impaction• Depends on particle stopping distance at the airway velocity


Response to Toxic Injury – Airway epithelium (carina)

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RESISTANCE THROUGH A TUBE

IS PROPORTIONAL TO1___________

(radius)4

Respiratory Response to Toxic Injury ‐ Airways

Diffuse large airway disease

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Airway Type Lining Cell Secretory Cell


Respiratory/ Terminal bronchiole




Respiratory Response to Toxic Injury ‐ Small Airways

Small Airways as Critical Sites for Damage

• Particle size of most microorganisms and irritant dusts (0.5‐2.0 m) effectively reach the bronchioalveolar junction (BAD) level and deposit

• No protective mucus layer (due to gas exchange function)

• Absence of ciliary apparatus

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Bronchiolar‐ alveolar duct junction (BADJ) common site for pathology



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Respiratory Response to Toxic Injury ‐ Alveoli

Terminal Airflow in the airspaces

red blood cell

type I pneumocyte

alveolar macrophage movingthrough alveolar pore of Kohn

fused basement membranesof capillary and type I pneumocyte

at areas of contactalveolar capillaries

interalveolar septumcontaining elastic and

collagen fibers


Alveolar Cell Types

Airway Type Lining Cell Secretory Cell


Terminal bronchiole




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Alveolar epithelium

• Type I cells ‐

– squamous, large surface area ‐ susceptible to damage

– maintain barrier to fluid loss, allow diffusion of gases,

– cannot divide

• Type II cells ‐

– cuboidal, lamellar bodies (surfactant), corners of alveoli

– divide to replace type I and II cells

– synthesizes surfactant, type IV collagen, PAF,

– aa metabolites

– modulate macrophage function

Respiratory Response to Toxic Injury – Alveoli

Retention and Clearance of Deposited Particles

Alveolar Region:

– No protective mucus layer due to gas exchange function

– Soluble particles

• Pass through membrane

into blood

– Insoluble particles

• Engulfed by alveolar macrophages

– Lymph nodes

– Mucociliary escalator


Alveolar macrophages

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Respiratory Response to Toxic Injury – Airspaces (Alveoli)

Retention and Clearance of Deposited ParticlesMacrophage accumulation


Alveolar inflammation


Alveolar edema

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Diffuse alveolar damage


Diffuse alveolar damage


Diffuse alveolar damage ‐ sequel

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Alveolar destruction ‐ emphysema

Response to Foreign Material

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Inhaled inert material


Alveolar fibrosis (Silica – 30 days)

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Respiratory Response to Toxic Injury

Collection / Inflation Procedures for Lung

I. Syringe technique

II. Constant pressure technique


Importance of Fixation


Syringe technique

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Constant pressure technique

– Advantages

• Reasonably inexpensive

• Excellent control over pressure

• Least amount of artifact

– Disadvantages

• Requires more time

• Availability of apparatus

Airway Macrophages

Air inflation/immersion fixationMigrating macrophages in situ

Intratracheal fixationMacrophages flushed from airway


Case Study

Macrophage aggregations in the lung

Adverse? Non‐adverse? Incidental?


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Variable terminology for macrophages in the lung

• alveolar foamy macrophages• alveolar histiocytosis• prominent alveolar macrophages• intra‐alveolar macrophages• foamy alveolar macrophages• increased macrophage aggregates• alveolar macrophage aggregation• macrophage aggregation, alveolar


Alveolar macrophages

Female High Dose

x5 x40


Rat 4 Week Tox Study

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Rat 4 Week Tox Study

Female Control

x5 x40


Foamy Macrophages (2)

• Rat 26‐week study (3, 30 and 300mg/kg/day), alveolar foamy macrophages in control rats (7/12 control males & 5/12 control females) in rats at the end of treatment period

• In males, foamy macrophages similar to concurrent controls, but at slight grade in a small proportion of animals with no clear dose response

• In females, an increased alveolar foamy macrophages in all treated groups. Some rats at slight grade & single rat at 300 mg/kg/day at moderate grade

• Recovery group: increased incidence in males & an increase in severity in females at 300 mg/kg/day at end of recovery phase

Male Female Dose (mg/kg/day) 0 3 30 300 0 3 30 300 Number Examined 12 12 10 11 12 12 12 12 Alveolar Foamy Macrophage/s

Minimal 7 6 5 6 5 6 5 7 Slight 0 2 3 2 0 3 3 2

Moderate 0 0 0 0 0 0 0 1 Total 7 8 8 8 5 9 8 10

End of dosing phase:

End of recovery phase:

Male Female Dose (mg/kg/day) 0 300 0 300 Number Examined 6 6 6 6 Alveolar Foamy Macrophage/s

Minimal 1 4 3 0 Slight 1 1 0 3 Total 2 5 3 3


Rat Foamy Macrophages –Historical Data

Incidence and Severity of Foamy Alveolar Macrophages in Lung from aCarcinogenicity Study in Rats

MALE FEMALE

Group No. 1 2 3 4 5 1 2 3 4 5

Group Designation C L LI HI H C L LI HI H

No. of Animals/Group 60 60 60 60 60 60 60 60 60 60

Lung

Foamy Alveolar Macrophages

Grade 1

Grade 2

Grade 3

Grade 4

Total

38

3

3

0

44

36

2

0

1

39

31

8

0

0

39

30

3

1

1

35

30

12

4

1

47

27

4

0

0

31

21

9

2

0

32

36

8

1

0

45

23

10

4

0

37

23

9

4

1

37

C = Control. L = Low. LI = Low intermediate. HI = High intermediate. H = High.

Grade 1 = Minimal.Grade 2 = Slight.Grade 3 = Moderate.Grade 4 = Moderately severe.

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Alveolar macrophage response

What is clinical & regulatory significance of foamy macrophages in lungs from oral tox studies in rat (4 and 26 wk) ?

“Prior to long term clinical trials, provide adequate justification that the finding is within spontaneous background and/or is not an adverse finding, or identify a NOAEL dose in the rat by conducting an additional toxicity study”.


Unique aspects of Respiratory System

• Tremendous surface area

• Highly elastic tissue

• Low tissue density

• Delicate fluid air interface

• Dual blood supply

• Multiple types of air conducting units

• Complexities of cell types and locations

• Species variations

•Back ups

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Differences ‐ rodent and human

Anatomical features Human Rodent

Pleura Thick Thin

Airway Size Small calibre Large calibre

Submucosal glands Abundant Rare

Respiratory bronchioles Present Absent

Clara cell Low numbers High numbers

Mucus cell Abundant Rare (serous)

Cardiac muscle in pulmonary veins

Absent Present

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Inflation Fixation

• Syringe technique

– Advantages

• Quick, Inexpensive, Practical, Convenient

– Disadvantages

• Risk of variation in pressure

• Risk of overinflation, alveolar rupture

scitp presentation27jul12 - toxpath.org concepts in target organ toxicologic pathology respiratory...

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