Humidified-warm CO2 reduces

peritoneal metastasis following

laparoscopy in a colorectal mouse

model

Dr Sandra Carpinteri

Peter MacCallum Cancer Centre

Melbourne, Australia

Dry-cold CO2 damages mesothelial cells

CO2

Mesothelial cells

Microvilli

Basal lamina

Submesothelial

connective tissue

Histology of Peritoneum

Dry

-co

ld C

O2

No C

O2

200x

200x

500x

No insufflation

Dry-cold insufflation

Humidified-warm insufflation

Mesothelium present

Mesothelial cells bulged up

Denuded basal lamina

Mesothelial cells bulged up

Basal lamina not exposed

Erikoglu et al. 2005

Humidified-warm CO2 reduces peritoneal damage

HumiGardTM Fisher and

Paykel Healthcare, NZ Denuded

To study peritoneal damage and inflammation following laparoscopic

insufflation with dry-cold or humidified-warm CO2 gas in the context of

colorectal cancer

Secondary Aim

Primary Aim

To investigate peritoneal metastasis in mice insufflated with either dry-cold

or humidified-warm CO2 gas

Intubate and connect to ventilator…

Abdominal Insufflation

Experimental Design

Dry-cold CO2 Humidified-warm CO2 No insufflation

Inject colon cancer cells into peritoneal cavity

CT26 cells

-Murine colorectal

carcinoma

-Modified to express

fluorescent protein

Harvest peritoneum

Aim One

Tumour burden

Aim Two

10 days 2 hours - 7 days

Damage and inflammation

Humidified-warm CO2 reduces metastatic tumour burden

M a c ro s c o p ic tu m o u r b u rd e n to th e p e r ito n e u m

fo llo w in g la p a ro s c o p ic C O 2 in s u ffla tio n

Nu

mb

er

of

tum

ou

rs

D r y-c o ld H u m id if ie d -w a r m N o in s u ff la t io n

0

2

4

6

8

C O 2 c o n d it io n s

*

Carpinteri et al. Annals of Surgery – under review

Port tumour

Humidified-warm CO2…

Reduced hypothermia during our mouse laparoscopic insufflation

Protected the peritoneum from damage compared to cold-dry CO2 insufflation

C O X -2

T im e o f h a rv e s t

H s

co

re

0

1

2

3

4

2  h 8  h 2 4  h 4 8  h 7  d

N o in s u ffla tio n

D ry -c o ld

H u m id ifie d -w a rm*

*

*

Dry-cold CO2…

Significantly increases peritoneal inflammation

following laparoscopic insufflation (measured

by COX-2 expression)

Secondary Outcomes

A v e ra g e te m p e ra tu re o v e r

th e 1 h o u r la p a ro s c o p ic p ro c e d u re

M in u te s

De

gre

es

0 1 0 2 0 3 0 4 0 5 0 6 0

0

3 4

3 6

3 8

**

*

5

3 6 .5

3 7 .5

N o in s u ffla tio n

D ry -c o ld

H u m id ifie d -w a rm

Summary

Conclusion One

Humidified-warm CO2 significantly reduces peritoneal metastasis.

Conclusion Two

It is easier to maintain normothermia using humidified-warm CO2 for

insufflation.

Insufflation with humidified-warm CO2 reduces peritoneal damage.

Dry cold CO2 enhances peritoneal inflammation following laparoscopic

insufflation.

Where to next?

Clinical trial using humidified-warm CO2 during cancer surgery.

Investigate the use of CO2 in an open-surgery colorectal cancer model.

Acknowledgments

Peter MacCallum Cancer Centre

Differentiation and Transcription Lab

Prof Rob Ramsay

Prof Craig Lynch (CRC surgeon)

Dr Maria-Pia Bernardi

Shienny Sampurno

Dr Markus Germann

Animal facility

Histology & Microscopy

Fisher & Paykel Healthcare

Michael Blackhurst

Jess Fogarin

Stephanie Marshall

Melbourne University

Dr Brenton Chambers

Baker IDI Heart and Diabetes Institute

Dr Xiao-Jun Du (Cardiac surgeon)

Dry-cold CO2 denudes basal lamina

Volz et al. 1999

- Mice insufflated 30 min, 6mmHg

- Peritoneum harvested 2 h

following procedure

710x

710x

1310x

Mesothelial cells have partially retracted, strongly

bulged up, and appear nearly spherical

Intercellular clefts and the underlying basal lamina

are clearly visible

Normal mesothelium

Dry-cold insufflation

Volz et al. 1999

Reducing inflammation using humidified-warm CO2

Dry-cold insufflation Humidified-warm insufflation

20x 20x

• Peritoneum harvested 8 hours following CO2 insufflation

• Immunohistochemistry inflammatory marker COX-2

C O X -2

T im e o f h a rv e s t

H s

co

re

0

1

2

3

4

2  h 8  h 2 4  h 4 8  h 7  d

N o in s u ffla tio n

D ry -c o ld

H u m id ifie d -w a rm*

*

*

200x

200x

500x

No insufflation

Dry-cold insufflation

Humidified-warm insufflation

Mesothelium present

Mesothelial cells bulged up

Denuded basal lamina

Mesothelial cells bulged up

Basal lamina not exposed

Erikoglu et al. 2005

Humidified-warm CO2 reduces peritoneal alterations

Erikoglu et al. 2005

- Rats insufflated 2 h, 10mmHg

- Peritoneum harvested 12 h

following procedure

HumiGardTM Fisher and Paykel Healthcare, NZ

Humidified-warm CO2 protects the peritoneal mesothelium

No insufflation Dry-cold Humidified-warm Absent

Short

Long

M ic ro v ill i

Ex

ten

t (%

)

2 h 8 h 2 4 h 4 8 h 7 d 2 h 8 h 2 4 h 4 8 h 7 d 2 h 8 h 2 4 h 4 8 h 7 d

0

5 0

1 0 0L o n g

S h o rt

A b s e n t

N o in s u f f la t io n D r y -c o ld C O 2 H u m id if ie d -w a r m C O 2

*

*#

^

N o in s u f f . V S D ry -c o ld

N o in s u f f . V S H u m id -w a rm

D ry -c o ld V S H u m id -w a rm

## ^

* ^

*#

* ^#^^*

*#

**

#

M ic ro v ill i

Ex

ten

t (%

)

2 h 8 h 2 4 h 4 8 h 7 d 2 h 8 h 2 4 h 4 8 h 7 d 2 h 8 h 2 4 h 4 8 h 7 d

0

5 0

1 0 0L o n g

S h o rt

A b s e n t

N o in s u f f la t io n D r y -c o ld C O 2 H u m id if ie d -w a r m C O 2

*

*#

^

N o in s u f f . V S D ry -c o ld

N o in s u f f . V S H u m id -w a rm

D ry -c o ld V S H u m id -w a rm

## ^

* ^

*#

* ^#^^*

*#

**

#

High flow = peritoneal damage

= cancer cell adhesion

Pilot trial – Flow rate: 1 L/min

Amended flow: 25-50 mL/min

Entry port

Exit port

Flow rate optimised for successful insufflation modelling human laparoscopy

F 4 /8 0

T im e o f h a rv e s t

H s

co

re

0

0

2

4

6

8

1 0N o in s u ffla tio n

D ry -c o ld

H u m id ifie d -w a rm

2  h 8  h 2 4  h 4 8  h 7  d

*

Enhanced peritoneal repair following dry-cold CO2 insufflation

Immunohistochemistry – Macrophage detection

Damage + cancer dissemination = metastasis

Laparoscopy Port insertion Port site tumour

Peritoneal metastasis

Humidified-warm CO2 maintains normothermia

during insufflation A v e ra g e te m p e ra tu re o v e r

th e 1 h o u r la p a ro s c o p ic p ro c e d u re

M in u te s

De

gre

es

0 1 0 2 0 3 0 4 0 5 0 6 0

0

3 4

3 6

3 8

**

*

5

3 6 .5

3 7 .5

N o in s u ffla tio n

D ry -c o ld

H u m id ifie d -w a rm

Carpinteri et al. – manuscript submitted

P < 0.02

Humidified-warm CO2 protects the peritoneal mesothelium

No insufflation Dry-cold Humidified-warm

Absent Short Long

Result

Examples of microvilli damage

Humidified-warm CO2 reduces damage and subsequently tumour adhesion

M e s o th e liu m

Ex

ten

t (%

)

2 h 8 h 2 4 h 4 8 h 7 d 2 h 8 h 2 4 h 4 8 h 7 d 2 h 8 h 2 4 h 4 8 h 7 d

0

5 0

1 0 0

N o d a m a g e

R o u n d e d /re tra c te d

N o in s u f f la t io n D r y -c o ld C O 2 H u m id if ie d -w a r m C O 2

*#

^

N o in s u f f . V S H u m id -w a rm

D ry -c o ld V S H u m id -w a rm

N o in s u f f . V S D ry -c o ld

* ^** ^*No insufflation

Dry-cold

Humidified-warm

C a n c e r c e ll a d h e s io n

Ex

ten

t (%

)

2 h 8 h 2 4 h 4 8 h 7 d 2 h 8 h 2 4 h 4 8 h 7 d 2 h 8 h 2 4 h 4 8 h 7 d

0

5 0

1 0 0N o a d h e s io n

A d h e s io n

N o in s u f f la t io n D r y -c o ld C O 2 H u m id if ie d -w a r m C O 2

*#

^

N o in s u f f . V S H u m id -w a rm

D ry -c o ld V S H u m id -w a rm

N o in s u f f . V S D ry -c o ld

* ^* **

^^* ^* ^

^

Dry-cold CO2 denudes basal lamina

710x

710x

1310x

Mesothelial cells have partially retracted, strongly

bulged up, and appear nearly spherical

Intercellular clefts and the underlying basal lamina

are clearly visible

Normal mesothelium

Dry-cold insufflation

Volz et al. 1999