Introduction Perinatal brain injury is a group of pathological conditions caused by exposure of the fetus to adverse factors in the antenatal period, during birth and in the first days after birth which results in various neurological disorders. Hypoxia- Ischaemia (HI – oxygen starvation), and inflammation (infectious agents) are the leading causes of severe damages to the vulnerable developing central nervous system of the perinate. The depletion of oxygen and glucose supply upon HI leads to impairments of energy resources and activation of necrotic and apoptotic circuits in neuronal cells, where mitochondria perform as a main hub of injury responses in the developing brain (Figure 1) (Hagberg et al., 2014). Inflammation, the other major cause of perinatal brain injuries, is induced by maternal or intrauterine fetal infection and leads to inflammation of the chorion/amniotic membranes, to activation of innate defensive cells response (microglia), which in turn enhances them to release several chemokines, pro-inflammatory cytokynes and free radicals causing detrimental effect to neurons (Dheen et al., 2007). Inflammation interrupts hemodynamic stability in the fetus; hence it is likely that hypoxic and inflammatory pathways interact to augment brain damage (Rees et al., 2011). Methylene Blue (MB), the first synthetic drug, has been applied in clinics for various diseases for more than a century (Ryou et al., 2015). In recent years, the application of MB as one of the cellular approaches for neurological disorders is increasing. MB has been shown to attenuate mitochondrial dysfunction under stress (Zhang et al., 2006), increase mitochondrial complex IV activity (Atamna et al., 2008; Poteet et al., 2012), MB has been reported to increase astrocyte energy storage by enhancing glucose metabolism and mitochondrial oxidative phosphorylation (Choudhury et al., 2015). Figure 2 depicts proposed mechanism of action of MB. Ameliorating mitochondrial dysfunction as a treatment of perinatal brain damage Aims • To study the effect of MB over neuronal progenitor, oligodendrocyte precursor and microglial cells’ survival during and after oxygen and glucose deprivation (OGD)-reoxygenation as a cell model of HI insult. •To determine how MB treatment can influence the mitochondrial metabolism in response to OGD insult. Methods During this project following cell lines were used (Figure 3 A-D): Figure 3. Cell lines used throughout the project. A. Neuronal progenitor cell line C17.2. B. Oligodendrocyte precursor CG4 cell line. C. Microglial BV2 cell line. D. Microglial N9 cell line. A summarised workflow of this project indicated in the following ( Figure 4 A-C). Results MB protects C17.2, CG4 cells from OGD induced cell death. In C17.2 and CG4 cells four hours of OGD and 24 hrs of reoxygenation caused around 50% cell loss in the MBctr (no MB treatment) group (Figure 5 A-D.). MB (0.5μM) treatment after OGD was able to significantly rescue the cells from injury caused by OGD for both cell lines (Figure 5 B,D.) Fig. 5. LDH assay data analysing cell viability of C17.2/CG4 cell lines upon HI. ( A). Exposure of C17.2 cells to OGD-reoxygenation caused approx. 40% cell loss and MB treatment during OGD did not caused any protective effect but increased the cell death, while (B) MB (0.5μM) treatment administered during reoxygenation significantly protected C17.2 cells from OGD-induced cell death. (C) Exposure of CG4 cells to OGD caused 50% cell death, MB treatment during OGD increased cell death, and (D) MB (0.5μM, 1μM) treatment after OGD indicated considerable protection from OGD. * p<0.05 significance to the CONTROL, # p<0.05 significance to the MB_ctr (No MB treatment). MB reduces the reactive oxygen species production level in C17.2 cell lines after OGD-induced cell death. After 4hr OGD +24 hr reoxygenation C17.2 cells were stained with CellRox® Deep Green reagent and fixed with 4% formaldehyde. CellRox® Green reagent is a dye which upon oxidation binds to DNA, and its signal localized in the cell’s nucleus and mitochondria. Results suggest that MB treatment during and after OGD suppressed the reactive oxygen species generation in C17.2 cells caused by OGD settings, (Figure 6 A,B). Figure 7 (A-D) demonstrates pictures imaged on EVOS microscopy system, and indicates the fluorogenic signal of CellRox® Green upon oxidation caused by OGD. Fig. 6. Reactive oxygen species production analysis using CellRox® Deep Geen reagent. (A) HI mimicking OGD settings caused significantly high amount of ROS production in variant with no MB treatment, and MB treatment during OGD inhibited the ROS production level almost by 50% with no statistical significance. (B) MB (0.1μM and 1μM) treatment during reoxygenation also suppressed ROS amount with statistical significance # p<0.05 to the MB_ctr. Fig. 7. Fluorescence-life time imaging microscopy of C17.2 cells stained with CellRox ® Deep Green at 24 hours after reoxygenation with or without MB treatment. The intensity of the fluorogenic signal of CellRox® Green dye is proportional to the amount of produced ROS in the cells upon OGD. (A) Control group demonstrated the lower intensity of the fluorogenic signal, whilst (B) 4hr OGD caused high production of ROS corresponding to saturated signal of CellRox® Green. (C) MB treatment during OGD (0.5μM) and (D) MB treatment during reoxygenation (0.5μM) indicating inhibitory effect of MB over ROS production. MB restores the energy production in CG4 cells after OGD insult. After 4hr OGD and 2hr reox. the ATP reagent (MitoToxGlo™) were introduced to CG4 cells and the luminescence was measured using GloMax multidetection system. Luminescence signal is proportional to the level of ATP present in the cells. Results demonstrated an increase of ATP levels in CG4 cells MB treated during/after OGD (Figure 8 A-B). Conclusion: The aims of the studies demonstrated here were to determine the potential protective effect of Methylene Blue on different brain cells upon Hypoxia-Ischaemia mimicking settings. From Figures 5,6 , 7 and 8 it can be concluded that: - MB protects C17, CG4 cells against transient OGD-induced cell death - MB reduces the ROS production level iC17.2 cell line after OGD by 50% - MB treatment restores the energy production in CG4 cells after OGD by increasing the ATP levels. In summary, the preliminary results obtained from studies on C17, CG4 cells suggest that MB has protective effect over these neuronal cells survival upon OGD-induced cell death. This protection can be attributed to decrease of the ROS production and restore of the ATP production in OGD treated cells. This potential protective feature of MB might provide a novel cellular approach to maintaining brain energy metabolism and improving the outcome after HI insult in perinatal brain. Akerke Bissenbay (BSc), Anton Kichev (PhD) Centre for Developing Brain, Perinatal Brain Injury Group, King’s College London Figure 1. Role of mitochondria in cell death in the immature brain . Calcium influx and accumulation in the cell causes mitochondrial swelling and mitochondrial permeabilization, releasing ROS and subsequently promoting apoptosis. Figure 2. Schematic representation of the proposed mechanism of action of the MB. MB minimizes the electron leakage by transferring electrons from complex I to cytochrome c bypassing complex II and III. A B C D A Figure 4. Time course and settings of experiments . A. Experiments with C17.2/CG4 cell lines on cell viability and ATP measurements. B. Microscopy of C17.2 cells. C. Experiments with microglial BV2 and N9 cell lines B C References: • Atamna H., Nguyen A., Schulz C., Boyle K., Newberry J., Kato H., Ames B.N. (2008). Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. The FASEB Journal. 22, 703-712. • Choudhury G.R., Winters A., Rich R.M., Ryou M.. (2015) Methylene Blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration. PLOS One. 1-14 • Hagberg H., Mallard C., Rousset C.I., Thornton C. (2014). Mitochondria: hub of injury responses in the developing brain. The Lancet. 13, 217-228. • Poteet E., Winters A., Yan L.J., Shufelt K., Green K.N., Simpkins J.W. (2012). Neuroprotective actions of methylene blue and its derivatives. PloS one. 7 (10). • Rees S., Harding R., Walker D. (2011). The biological basis of injury and neuroprotection in the fetal and neonatal brain. International Journal of Developmental Neuroscience. 29, 551-563. • Ryou M.G., Choudhury G.R., Li W., Winters A., Yuan F., Liu R., Yang S.H. (2015). Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress. Neuroscience. 301, 193-203. A B * C D A B Future studies: - Further studies with C17.2, CG4 cell lines (ROS analysis, ATP measurements) - Studies with microglial BV2/N9 cell lines to determine the protective effect of MB on them upon inflammatory conditions. - Determine the type of cell death (necrotic/apoptotic) after OGD and determine the activated apoptotic pathways. -Confirm the protective effect of MB with primary cell lines. -- Animal studies ( brain sectioning, immunohistochemistry). # Absorbance (450nm-750nm) C17.2 cells Absorbance (450nm/750nm) C17.2 cells OGD-reoxygenation OGD-reoxygenation Absorbance (450nm/750nm) CG4 cells Absorbance (450nm/750nm) CG4 cells Signal intensity ROS production level Signal intensity ROS production level OGD-reoxygenation OGD-reoxygenation A B C D Luminescence CG4 cells – 4hr OGD + 2hr reox Luminescence CG4 cells- 4hr OGD + 2hr reox # # # # # A B Fig. 8. Quantitative analysis of ATP generation in MB treated CG4 cells at 2 hrs of reoxygenation. (A) and (B) transient 4 hr OGD caused around 60% reduction in ATP production, while MB administration during OGD (A) as well as MB treatment during 2hr reoxygenation (B) was able considerably restore the energy production levels in CG4 cells.
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Introduction Perinatal brain injury is a group of pathological conditions caused by exposure of the fetus to adverse factors in the
antenatal period, during birth and in the first days after birth which results in various neurological disorders. Hypoxia-
Ischaemia (HI – oxygen starvation), and inflammation (infectious agents) are the leading causes of severe damages to
the vulnerable developing central nervous system of the perinate.
The depletion of oxygen and glucose supply upon HI leads to impairments of energy resources and activation of necrotic
and apoptotic circuits in neuronal cells, where mitochondria perform as a main hub of injury responses in the
developing brain (Figure 1) (Hagberg et al., 2014).
Inflammation, the other major cause of perinatal brain injuries, is induced by maternal or intrauterine fetal infection and
leads to inflammation of the chorion/amniotic membranes, to activation of innate defensive cells response (microglia),
which in turn enhances them to release several chemokines, pro-inflammatory cytokynes and free radicals causing
detrimental effect to neurons (Dheen et al., 2007). Inflammation interrupts hemodynamic stability in the fetus; hence it is
likely that hypoxic and inflammatory pathways interact to augment brain damage (Rees et al., 2011).
Methylene Blue (MB), the first synthetic drug, has been applied in clinics for various diseases for more than a century
(Ryou et al., 2015). In recent years, the application of MB as one of the cellular approaches for neurological disorders is
increasing. MB has been shown to attenuate mitochondrial dysfunction under stress (Zhang et al., 2006), increase
mitochondrial complex IV activity (Atamna et al., 2008; Poteet et al., 2012), MB has been reported to increase astrocyte
energy storage by enhancing glucose metabolism and mitochondrial oxidative phosphorylation (Choudhury et al., 2015).
Figure 2 depicts proposed mechanism of action of MB.
Ameliorating mitochondrial dysfunction as a treatment
of perinatal brain damage
Aims • To study the effect of MB over neuronal progenitor, oligodendrocyte precursor and microglial cells’
survival during and after oxygen and glucose deprivation (OGD)-reoxygenation as a cell model of HI
insult.
•To determine how MB treatment can influence the mitochondrial metabolism in response to OGD
insult.
Methods
During this project following cell lines were used (Figure 3 A-D):
Figure 3. Cell lines used throughout the project. A. Neuronal progenitor cell line C17.2. B. Oligodendrocyte precursor CG4 cell
line. C. Microglial BV2 cell line. D. Microglial N9 cell line.
A summarised workflow of this project indicated in the following (Figure 4 A-C).
Results
MB protects C17.2, CG4 cells from OGD induced cell death. In C17.2 and CG4 cells four hours of OGD and 24 hrs of reoxygenation caused around 50% cell loss in the MBctr (no MB treatment) group (Figure 5 A-D.). MB (0.5µM) treatment after OGD was able to significantly rescue the cells from injury caused by OGD for both cell lines (Figure 5 B,D.)
Fig. 5. LDH assay data analysing cell viability of C17.2/CG4 cell lines upon HI. (A). Exposure of C17.2 cells to OGD-reoxygenation caused approx. 40% cell loss and MB treatment during OGD did not caused any protective effect but increased the cell death, while (B) MB (0.5µM) treatment administered during reoxygenation significantly protected C17.2 cells from OGD-induced cell death. (C) Exposure of CG4 cells to OGD caused 50% cell death, MB treatment during OGD increased cell death, and (D) MB (0.5µM, 1µM) treatment after OGD indicated considerable protection from OGD. * p<0.05 significance to the CONTROL, # p<0.05 significance to the MB_ctr (No MB treatment).
MB reduces the reactive oxygen species production level in C17.2 cell lines after OGD-induced cell death. After 4hr OGD +24 hr reoxygenation C17.2 cells were stained with CellRox® Deep Green reagent and fixed with 4% formaldehyde. CellRox® Green reagent is a dye which upon oxidation binds to DNA, and its signal localized in the cell’s nucleus and mitochondria. Results suggest that MB treatment during and after OGD suppressed the reactive oxygen species generation in C17.2 cells caused by OGD settings, (Figure 6 A,B). Figure 7 (A-D) demonstrates pictures imaged on EVOS microscopy system, and indicates the fluorogenic signal of CellRox® Green upon oxidation caused by OGD.
Fig. 6. Reactive oxygen species production analysis using CellRox® Deep Geen reagent. (A) HI mimicking OGD settings caused significantly high amount of ROS production in variant with no MB treatment, and MB treatment during OGD inhibited the ROS production level almost by 50% with no statistical significance. (B) MB (0.1µM and 1µM) treatment during reoxygenation also suppressed ROS amount with statistical significance # p<0.05 to the MB_ctr.
Fig. 7. Fluorescence-life time imaging microscopy of C17.2 cells stained with CellRox ® Deep Green at 24 hours after reoxygenation with or without MB treatment. The intensity of the fluorogenic signal of CellRox® Green dye is proportional to the amount of produced ROS in the cells upon OGD. (A) Control group demonstrated the lower intensity of the fluorogenic signal, whilst (B) 4hr OGD caused high production of ROS corresponding to saturated signal of CellRox® Green. (C) MB treatment during OGD (0.5µM) and (D) MB treatment during reoxygenation (0.5µM) indicating inhibitory effect of MB over ROS production.
MB restores the energy production in CG4 cells after OGD insult. After 4hr OGD and 2hr reox. the
ATP reagent (MitoToxGlo™) were introduced to CG4 cells and the luminescence was measured using GloMax multidetection system. Luminescence signal is proportional to the level of ATP present in the cells. Results demonstrated an increase of ATP levels in CG4 cells MB treated during/after OGD (Figure 8 A-B).
Conclusion: The aims of the studies demonstrated here were to determine the potential
protective effect of Methylene Blue on different brain cells upon Hypoxia-Ischaemia mimicking
settings. From Figures 5,6 , 7 and 8 it can be concluded that:
- MB protects C17, CG4 cells against transient OGD-induced cell death
- MB reduces the ROS production level iC17.2 cell line after OGD by 50%
- MB treatment restores the energy production in CG4 cells after OGD by increasing the ATP
levels.
In summary, the preliminary results obtained from studies on C17, CG4 cells suggest that MB has
protective effect over these neuronal cells survival upon OGD-induced cell death. This protection
can be attributed to decrease of the ROS production and restore of the ATP production in OGD
treated cells. This potential protective feature of MB might provide a novel cellular approach to
maintaining brain energy metabolism and improving the outcome after HI insult in perinatal
brain.
Akerke Bissenbay (BSc), Anton Kichev (PhD)
Centre for Developing Brain, Perinatal Brain Injury Group, King’s College
London
Figure 1. Role of mitochondria in cell death in the immature brain . Calcium influx and accumulation in
the cell causes mitochondrial swelling and mitochondrial permeabilization, releasing ROS and subsequently
promoting apoptosis.
Figure 2. Schematic representation of the proposed mechanism of action of the MB. MB minimizes the
electron leakage by transferring electrons from complex I to cytochrome c bypassing complex II and III.
A B C D
A
Figure 4. Time course and settings of experiments. A. Experiments with C17.2/CG4 cell lines on cell viability and ATP measurements. B.
Microscopy of C17.2 cells. C. Experiments with microglial BV2 and N9 cell lines
B
C
References: • Atamna H., Nguyen A., Schulz C., Boyle K., Newberry J., Kato H., Ames B.N. (2008). Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. The FASEB
Journal. 22, 703-712.
• Choudhury G.R., Winters A., Rich R.M., Ryou M.. (2015) Methylene Blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration. PLOS One. 1-14
• Hagberg H., Mallard C., Rousset C.I., Thornton C. (2014). Mitochondria: hub of injury responses in the developing brain. The Lancet. 13, 217-228.
• Poteet E., Winters A., Yan L.J., Shufelt K., Green K.N., Simpkins J.W. (2012). Neuroprotective actions of methylene blue and its derivatives. PloS one. 7 (10).
• Rees S., Harding R., Walker D. (2011). The biological basis of injury and neuroprotection in the fetal and neonatal brain. International Journal of Developmental Neuroscience. 29, 551-563.
• Ryou M.G., Choudhury G.R., Li W., Winters A., Yuan F., Liu R., Yang S.H. (2015). Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress. Neuroscience. 301,
193-203.
A B
*
C D
A B
Future studies: - Further studies with C17.2, CG4 cell lines (ROS analysis, ATP measurements)
- Studies with microglial BV2/N9 cell lines to determine the protective effect of MB on them upon
inflammatory conditions.
- Determine the type of cell death (necrotic/apoptotic) after OGD and determine the activated
apoptotic pathways.
-Confirm the protective effect of MB with primary cell lines.
-- Animal studies ( brain sectioning, immunohistochemistry).
#
Ab
sorb
ance
(45
0n
m-7
50
nm
)
C17.2 cells
Ab
sorb
ance
(45
0n
m/7
50
nm
) C17.2 cells OGD-reoxygenation OGD-reoxygenation
Ab
sorb
ance
(45
0n
m/7
50n
m)
CG4 cells
Ab
sorb
ance
(45
0n
m/7
50n
m)
CG4 cells
Sign
al in
ten
sity
ROS production level
Sign
al in
ten
sity
ROS production level
OGD-reoxygenation OGD-reoxygenation
A B C D
Lum
ines
cen
ce
CG4 cells – 4hr OGD + 2hr reox
Lum
ines
cen
ce
CG4 cells- 4hr OGD + 2hr reox
#
#
#
#
#
A B
Fig. 8. Quantitative analysis of ATP generation in MB treated CG4 cells at 2 hrs of reoxygenation. (A) and (B) transient 4 hr OGD caused around 60%
reduction in ATP production, while MB administration during OGD (A) as well as MB treatment during 2hr reoxygenation (B) was able considerably restore
the energy production levels in CG4 cells.
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