Humidified-warm CO2 reduces peritoneal metastasis ... · reduces peritoneal metastasis following...
Transcript of Humidified-warm CO2 reduces peritoneal metastasis ... · reduces peritoneal metastasis following...
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
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
* ^* **
^^* ^* ^
^