Francisco Espejo Porras Final report - IBRO-InEurope Short Stay Grants Program

During my visit of four weeks working in the laboratory of Dr. Anke Witting I have learned to
work with glial cell primary cultures to study how cannabinoids can modulate inflammatory
response from activated glial cells with LPS. This is important due to the fact that glial
inflammation is a critical factor in ALS and it is a key element I wish to develop during my thesis
project.
Our first strategy was to check how the use of different cannabinoids agonists could modulate
the glial. ACEA and HU-308 were chosen, like CB1 and CB2 agonist respectively. It is well
known that CB1 agonists are neuroprotective and that when the glia are activated, it displays
an increase of CB2 receptor. In general there are two possibilities to study the effects of
cannabinoids treatment in culture cells. First we can add the cannabinoid before the treatment
with LPS (activator of inflammatory response) or after the activation. We have explored the
two options in these preliminary studies.
For my first week, the technician had made some preparations from glia. This first time we did
not have enough microglia cells to cultivate in plates, for this reason we started to work with
astrocytes. We checked different concentrations of cannabinoids to treat the LPS addition.
They also prepared crossings to obtain pups to do the glia preparation in this week. I trained to
obtain the preparations since the first step.
In a brief resume, during the stage P1-4 the pups are sacrificed and the brain is removed. After,
the meninges must be removed (it is possible that it would be the more difficult step) to avoid
any contamination from fibroblast. After that, through several steps using tripsine and
DNAasas, a glial homogeneizate is obtained and is cultivated in a culture flask. It is necessary to
wait 5-6 days to wash and change the medium. And another 3 days to do the first shake of the
microglia. This step you can repeat two more times after 3-4 days (you always check how are
the microglia under the microscope) or you can decide to work with astrocytes.
In these sense, during my second and third week I spent my time carrying out cultures cell of
astrocytes and microglia. And in my final week, I processed all the samples by ELISA kits in
order to measure the effects of treatments in the cytokine levels (TNF and IL6) of both
microglia and astrocytes culture cells.
Although I had planned work with SOD1 animals too, it was not possible because we could not
obtain an enough amount of pups to perform the culture properly. However, I have worked
with non-transgenic animals and have completed my learning period.

Results
The cytokine analysis from microglia cells was made from the 96 w/p cultures, but in the case
of astrocytes was used the cultures from 6 w/p. The following results do not show any
statistical study because they are only the result from one experiment (I hope to repeat it now
in my lab). At the same time, each valor from the different groups is really the average of two
wells from the same plate. I have normalized all the data respect to control group to facilitate
comparison between experiments.
In all the case I used 1g/ml of LPS during 24 hours or 48 hours.

- Microglia

ACEA TNF 24h

75000

55000

35000

15000
200
150
100
50
0

Ct
Ct
Ct
Ct
EtOH ACEA
10M 1M 0.1M

LPS
Ct

ACEA
ACEA
10M 1M 0.1M 10M 1M 0.1M
1 hour before

1 hour after

Only when the highest concentration of ACEA (10M) is used and before the addition of LPS
does a decrease of TNF level show.

ACEA TNF 48h

65000

45000

25000

5000

100
50
0

Ct
Ct
EtOH ACEA
10M

Ct

LPS

1M

Ct

ACEA
10M

1M 0.1M

1 hour before

ACEA
10M

1M 0.1M

1 hour after

But with a treatment during 48 hours of LPS, ACEA appears be effective inclusive after the LPS
addition being more potent when is used before.
HU-308 TNF 24h
75000

55000

35000

15000
200
150
100
50
0

Ct
Ct
Ct
Ct
EtOH HU-308
1M 0.1M 10nM

LPS
Ct

Hu-308
HU-308
1M 0.1M 10nM 1M 0.1M 10nM
1 hour before

1 hour after

In the case that the CB2 agonist is used, it only seems to be effective when it is added (and
only the highest concentration) after the LPS stimuli (in a 24 hours treatment).

HU-308 TNF 48h

65000

45000

25000

5000
100
50
0

Ct
Ct
Ct
EtOH HU-308
1M 0.1M

LPS
Ct

HU-308
HU-308
1M 0.1M 10nM
1M 0.1M 10nM
1 hour before

1 hour after

If the treatment is for 48 hours, the presence of HU-308 could also be effective before the LPS
stimuli, but after seems to be more effective.
Seeing that only ACEA 10M and HU-308 1M could have effect, to measure the IL6 levels I
focused in these concentrations.
IL6 24h
20000

15000

10000

5000

400

200

Ct
EtOH

Ct
Ct
ACEA HU-308

LPS
Ct

ACEA HU-308 ACEA HU-308

1 hour before
1 hour after

The levels of IL6 could be decreased in the LPS control group in both cases. The pretreatment
is most effective 1 hour before and using the CB1 agonist.

IL6 48h
20000

15000

10000

5000

750
500
250
0

Ct
EtOH

Ct
Ct
ACEA HU-308

LPS
Ct

ACEA HU-308 ACEA HU-308

1 hour before
1 hour after

But it is surprising that if the LPS stimuli it is for 48 hours, only the pretreatment with ACEA
obtain an IL6 level decreased.

- Astrocytes

ACEA TNF 24h

150

ACEA TNF 48h

150

100

100

50

50

Ct
EtOH

Ct
ACEA
10M

LPS
Ct

ACEA
1h bef

1h aft

16h aft

Ct
Ct
EtOH ACEA
10M

LPS
Ct

ACEA
1h bef 1h aft 24h aft 40h aft

The levels of TNF only are decreased when you add de CB1 agonist 1 hour before the LPS.

HU-308 TNF 24h

150

HU-308 TNF 48h

150

100

100

50

50

Ct
Ct
EtOH HU-308
1M

LPS
Ct

HU-308
1h bef

1h aft

Ct
Ct
LPS
EtOH HU-308
Ct HU-308
1M
1h bef 1h aft 24h aft 40h aft

16h aft

HU-308 only seems to be a little effective when the LPS stimulii during 48 hours.
In both experiments a LPS stimuli was necessary to detect any TNF level.
ACEA IL6 48h

ACEA IL6 24h

400

600

300

400
200

200
100

Ct
EtOH

Ct
ACEA
10M

LPS
Ct

ACEA
1h bef

1h aft

Ct
Ct
EtOH ACEA
10M

16h aft

LPS
Ct

ACEA
1h bef 1h aft 24h aft 40h aft

Respect the IL6 level, there is only a decrease when ACEA is added 1 hour before the LPS.

HU-308 IL6 24h

600

1000

HU-308 IL6 48h

800
400

600

400
200

200
0

Ct
Ct
EtOH HU-308
1M

LPS
Ct

HU-308
1h bef

1h aft

16h aft

Ct
Ct
LPS
EtOH HU-308
Ct HU-308
1M
1h bef 1h aft 24h aft 40h aft

It happened equal with HU-308, that only 1 hour before the LPS reach to decrease the IL6
level.
Conclusions
At first, it is necessary to consider that all these results have to be repeated and confirmed but
there are a few ideas that we could have. It is clear that the LPS stimuli affect more microglia
than astrocytes referring to TNF and IL6 levels. If we are focusing on microglia, we have had
in general the protective effect using a CB1 agonist like ACEA is, when it was added before the
LPS stimuli. Although when HU-308 is used, it seems to have more effect after the LPS stimuli.
This effect could be the opposite in astrocytes because there is a little trend to increment the
level of cytokines when we add HU-308 after the LPS stimuli.

Discussion
It is possible that ACEA and HU-308 have a low activity in astrocytes due to the fact that they
have a low presence of TLR4 receptors. Moreover, the liberation of TNF and IL6 is a principal
role of microglia and not of astrocytes. In one hand, the results could confirm the
neuroprotector role of CB1 agonist because ACEA 1 hour before preventing the inflammation.
But in the other hand, it is more interesting to see the results with HU-308 due to the different
implications that could occur. It is known that the CB2 receptors can increase once that the LPS
stimuli is done. In addition, the activation via CB2 could allow a microglial change since M1
state (the classical pro-inflammatory pathway) to M2 (the alternative pathway is related with
neuroprotection).
These are preliminary data, but once we could confirm the idea is to repeat it with mutant
SOD1 or TDP43 glia. Logically, it would be necessary to do studies about morphology or what
kind of glial marks are expressed in these cultures after the cannabinoid treatment. Finally it
would be interesting to do experiments with conditioned mediums in neurons.

Acknowledgments
I want to thank the IBRO In Europe Short Stay Grants Program for allowing me to enjoy this
experience in Ulm. I also want to thank specially Dr. Anke Witting for all the help she gave me.
She has been a great supervisor and she took a lot of care of my work there. With any
question, any requirement, she always made time for me and was very kind.