In Situ Imaging of Cysteine in the Brains of Mice with Epilepsy by a Near-Infrared Emissive Fluorescent Probe

Article abstract of DOI:10.1021/acs.analchem.9b05211

Epilepsy is characterized by oxidative stress in the brain. As the crucial reductive biothiol, cysteine (Cys) directly regulates the occurrence of oxidative stress in the brain. Observations suggest that the decreased cysteine in plasma could potentially

Full text of DOI:10.1021/acs.analchem.9b05211

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Article
In-situ Imaging of Cysteine in the Brains of Mice with
Epilepsy by a Near-infrared Emissive Fluorescent Probe
Songjiao Li, Dan Song, Weijing Huang, Zhen Li, and Zhihong Liu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b05211 • Publication Date (Web): 06 Jan 2020
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Analytical Chemistry
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In-situ Imaging of Cysteine in the Brains of Mice with Epilepsy by a
Near-infrared Emissive Fluorescent Probe
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Songjiao Li, Dan Song, Weijing Huang, Zhen Li*, Zhihong Liu
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and
Molecular Sciences, Wuhan University, Wuhan, Hubei 430072, China.
ABSTRACT: Epilepsy is characterized by oxidative stress in the brain. As the crucial reductive biothiol, cysteine (Cys) directly
regulates the occurrence of oxidative stress in the brain. Observations suggest that the decreased cysteine in plasma could
potentially serve as a redox biomarker in temporal lobe epilepsy. However, due to the complexity of the brain and lack of
appropriate in-situ detecting tools, the concentration change and regulation of Cys in epileptic brains remain unclear. Here, we
report a near-infrared imaging probe (named Mito-CP) for tracking endogenous Cys in brains of pentylenetetrazole (PTZ)-induced
epileptic seizures with high sensitivity and selectivity. Using this probe, we achieved an in-situ visualization of the increased Cys in
PC12 cells under dithiothreitol stimulation. In addition, Mito-CP was able to real-time monitor Cys fluctuation in
lipopolysaccharide-mediated oxidative stress in zebrafish. Ultimately, we directly visualized the precipitous reduction of Cys in
epileptic brains for the first time. Mito-CP also revealed the fluctuation of Cys in epileptic brains during the treatment by an
antiepileptic drug, curcumin. This work provides an effective tool for Cys imaging in brains and will help to expand the
understanding of the pathogenesis of epilepsy.
Epilepsy is a brain disorder affecting over 50 million people
worldwide and is associated with increased mortality.1,2
Peripheral biomarkers for epilepsy are notably absent from the
penetration, higher spatial-temporal resolution and less
photodamage to biological specimen.13-16 Recently, some
fluorescence probes have been reported for Cys imaging in
living cells, zebrafish, and the abdomens of mice.17-23 However,
probes for in vivo imaging of Cys in brains has rarely been
reported.24
tools available to inform its diagnosis and treatment.3
A
growing body of work indicates that epilepsy is characterized
by oxidative damage to relevant biological components.4
Above all, mitochondrial oxidative stress and dysfunction are
emerging as key factors that not only result from seizures but
may also contribute to epileptogenesis. Mitochondria are the
main production source of reactive oxygen species (ROS), and
their oxidative damage is a crucial factor in cell death. In order
to avoid oxidative stress damage, mitochondria always have a
significant number of free radical scavengers, such as cysteine
(Cys), which acts as a vital antioxidant reservoir to regulate
ROS homeostasis and exerts cytoprotective effect against
oxidative damage. Therefore, defining the changes and
regulations of Cys is essential for a better understanding of the
molecular mechanism underlying epilepsy.5-7 Whereas current
studies on Cys in epilepsy mainly were implemented in
plasma.8 The existing methods cannot reflect Cys level in the
brain, especially, they are unable to track native Cys in real-
time with spatial resolution during epilepsy. Hence, to explore
definitive connections of Cys with epilepsy, it is demanded to
develop new tools for in-situ detection of Cys in the brain.
Herein, we proposed a novel mitochondria-targeting NIR
fluorescent probe (Mito-CP) for in-situ detection of Cys in the
brain. (Scheme 1). Mito-CP adopted Mito-Q as the
fluorophore and acrylate as the recognition site for Cys. Mito-
Q contains an N, N-dimethylamino moiety as an electron
donor and a quinoline cation as an electron acceptor. The
strong electron push-pull system causes an intramolecular
charge transfer (ICT) effect and a Stokes shift of ~260 nm.
Such a large Stokes shift can greatly improve the sensitivity by
reducing the overlap of excitation and emission bands. The
selection of acrylate as the recognition site was to make the
probe highly specific to Cys. As known, distinguishing Cys
from Hcy and GSH in physiological environments is quite
challenging, due to their similar structures and chemical
properties. It has been shown that some acrylate-
functionalized probes are prone to be more reactive toward
Cys over Hcy and GSH.25,26 Upon a conjugate addition-
cyclization reaction of Cys with acrylate and subsequent 1,6-
elimination of p-hydroxybenzyl moiety, bright fluorescence of
the probe at 700 nm is lighted up. More importantly, Mito-CP
possesses efficient blood-brain barrier (BBB) penetrability,
which endows the probe with the ability to rapidly enter the
brain after intravenous injection and map Cys in the brain. The
capability of Mito-CP to detect the variation of Cys, either
externally added or generated upon oxidative stress, was
demonstrated in varying organisms, including cultured cells,
zebrafish, and mice. It was successfully applied to visualize
Fluorescence imaging employing small-molecule probes
has emerged as a desirable and indispensable tool for
interrogating intact living samples.9-12 Due to the obvious
technical and practical advantages of good membrane
permeability, high sensitivity, and operational simplicity,
fluorescence probes are attracting increasing attention in life
science fields. Particularly, NIR excitable probes are
considered the most appropriate for brain imaging because
they provide a higher signal-to-background ratio, deeper tissue
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the fluctuation of Cys in mice brains induced by epileptic
seizures and treated with antiepileptic drugs. As far as we
know, this is the first in-situ study on the relationship between
Cys and epilepsy in brains with a molecular probe.
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and extracted with ethyl acetate for three times. Combined
organic layers were washed by saturated sodium thiosulfate
solution and dried by anhydrous Na2SO4. Then the solvent was
removed under reduced pressure and the residue was purified
by silica gel column chromatography to give the desired
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product 150 mg. Yield 75%. H NMR (400 MHz, CDCl3,
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Figure S1) δ 8.87 (d, J = 4.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H),
8.13 (d, J = 8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.61-7.52 (m,
5H), 7.48 (s, 1H), 7.12 (s, 2H), 6.74 (d, J = 8.0 Hz, 2H), 3.01
(s, 6H). 13C NMR (101 MHz, CDCl3, Figure S2) δ 150.3,
150.1, 148.8, 146.3, 142.7, 139.2, 130.1, 129.9, 129.3, 128.2,
126.8, 126.4, 126.2, 123.4, 122.1, 121.4, 120.3, 116.2, 112.4,
40.4. HRMS (Figure S3) m/z: calcd for C23H20N2S [M]+:
357.142, found: 357.142.
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Synthesis of Probe Mito-CP. A solution of compound 2
(100.0 mg, 0.42 mmol) and Mito-Q (150.0 mg, 0.42 mmol)
were mixed in acetonitrile (5 mL) and then piperidine (0.05
mL) was added to the solution. The reaction mixture was
refluxed with stirring. After the reaction was completed, the
reaction mixture was cooled to room temperature and solvent
was removed under reduced pressure. The resulting residue
was purified by column chromatography to give the desired
Scheme 1. Chemical structure of Mito-CP and proposed
reaction mechanisms of Mito-CP toward Cys.
EXPERIMENTAL SECTION
1
product 133 mg. Yield 53%. H NMR (400 MHz, DMSO,
Synthesis of Compounds. A synthetic route for compound
Mito-CP from commercially available compounds was
provided and depicted in Scheme 2. Compounds 1, 2 and 3
were synthesized according to references.27,28
Figure S4) δ 9.51 (d, J = 4.0 Hz, 1H), 8.98 (d, J = 8.0 Hz, 1H),
8.52 (t, J = 8.0 Hz, 2H), 8.40 (t, J = 12.0 Hz, 1H), 8.15 (t, J =
8.0 Hz, 1H), 7.96 (t, J = 8.0 Hz, 1H), 7.91 (d, J = 16.0 Hz, 1H),
7.76 (d, J = 4.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.53 (d, J =
4.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H),
6.80 (d, J = 8.0 Hz, 2H), 6.53 (d, J = 16.0 Hz, 1H), 6.42 (dd, J
= 8.0 Hz, 1H), 6.24 (s, 2H), 6.16 (d, J = 8.0 Hz, 1H), 3.00 (s,
6H). 13C NMR (101 MHz, DMSO, Figure S5) δ 164.5, 153.8,
153.6, 151.2, 147.9, 138.4, 138.1, 137.6, 135.9, 135.6, 134.6,
132.7, 129.4, 128.9, 127.9, 127.4, 126.9, 123.6, 123.4, 123.2,
122.9, 116.7, 116.1, 112.8, 58.7, 51.8. HRMS (Figure S6) m/z:
calcd for C33H29N2O2S [M]+: 517.194, found: 517.193.
Synthesis of Mito-Q. 4-methyl quinoline (160.0 mg, 1.12
mmol) was dissolved in dry DMF (10 mL) and then benzoyl
chloride (80.0 mg, 0.56 mmol) was added. The resultant
mixture was stirred for 20 minutes at room temperature. After
that, Compound 3 (130.0 mg, 0.56 mmol) was added and the
solution was refluxed for another 5 hours at 160 °C. When the
reaction was completed according to TLC, the reaction
mixture was cooled to room temperature, diluted with H2O
Scheme 2. Synthetic route of the probe Mito-CP.
increased, the absorption peak at 560 nm decreased gradually
combined with the emerging of a new absorption peak at 440
nm, which acts as the excitation wavelength of the reaction
product (the fluorophore). The fluorescence titration exhibited
a maximum enhancement of 12-fold at 100 μM Cys. In
addition, a good linearity was obtained over the concentration
range of 0.05-10.0 μM for Cys. The regression equation was y
= 160.89 + 88.307 × [Cys] with a linear coefficient R2 of
0.993. The limit of detection (LOD, S/N = 3) was calculated as
20.8 nM (Figure 1c), standing for a quite high sensitivity for
Cys among all the reported fluorescence probes.
RESULTS AND DISCUSSION
Sensing Properties of Mito-CP toward Cys. The response
of Mito-CP to Cys was first investigated in aqueous solutions.
Upon the addition of Cys, an increase of fluorescence intensity
was observed at 700 nm (Figure 1a). It is worth noting the
remarkable Stokes shift of 260 nm, which is larger than that of
most NIR emissive fluorescence probes and is in favor of
signal/noise ratio of imaging because of the reduction of cross-
talk between excitation and emission. Moreover, the UV-vis
absorption spectra of Mito-CP with different concentrations of
Cys were recorded (Figure 1b). With the Cys amount
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Figure 1. (a) Fluorescence spectra of Mito-CP (10 μM) in the presence of different concentrations of Cys (0-150.0 μM); (b)
absorption spectra of Mito-CP (10 μM) in the presence of different concentrations of Cys (0-10.0 μM); (c) linear relationship
between fluorescence intensity at 700 nm and concentrations of Cys; (d) fluorescence spectral changes of Mito-CP (10 μM) against
time in the presence of Cys (100 μM) in DMSO / PBS buffer (3:7, v/v, pH 7.4) at 37 °C; (e) Time-dependent response of Mito-CP
(10 μM) to Cys (100 μM), Hcy (100 μM) and GSH (10 μM), respectively; (f) Fluorescence responses of Mito-CP (10 μM) upon
addition of amino acids (1. Ala, 2. Glu, 3. Arg, 4. Ser, 5. Lys, 6. Asp, 7. Gly, 8. Leu, 9. Ile, 10. Glu, 11. Tyr, 12. His, 13. Trp, 14.
Thr, 15. Phe, 16. Asn, 17. Met, and 18. Val, 100 μM each) and biothiols (19. GSH, 100 μM; 20. Hcy, 100 μM; 21. Cys, 100 μM) in
DMSO / PBS buffer (3:7, v/v, pH 7.4) at 37 °C.
Next, the effect of pH on the fluorescence intensity of the
probe in absence and presence of Cys was examined. As
shown in Figure S7, the probe possessed optimal fluorescence
response toward Cys in the physiological pH range (7.0-8.0).
The time course studies showed that the fluorescence intensity
of the reaction plateaued in approximately 15 min and
remained virtually unchanged (Figure 1d). Next, we assessed
the specificity of Mito-CP to Cys over other amino acids and
thiols (Hcy and GSH) with similar structures. The time-
dependent fluorescence responses of probe to the thiols with
the same concentration were then monitored. As seen in
Figure 1e, as compared to the other thiols, the reaction
between Cys and Mito-CP exhibited significantly higher
reaction speed and higher fluorescence intensity at a steady-
state, suggesting the probe can be used to effectively
discriminate Cys from Hcy and GSH. Principally, the high
selectivity of probe Mito-CP for Cys over other thiols can be
attributed to the kinetic rate of the intramolecular cyclization
reactions. The intramolecular cyclization reaction is kinetically
favored for Cys to form a seven-membered ring compared to
that of Hcy which forms an eight-membered ring. The
intramolecular cyclization for GSH is hindered by the
bulkiness of its tripeptide; as a result, only the conjugated
thioether can be generated. Moreover, other amino acids
without thiol did not induce any detectable fluorescence
responses (Figure 1f). Other interfering substances such as
H2S, metal ions (K+, Na+, Ca2+, Mg2+), H2O2, HClO were also
investigated. As shown in Figure S8, no response was
observed.
properties of Mito-CP in response to Cys, theoretic calculation
by density functional theory (DFT) with the B3LYP/6-31G (d)
method was conducted using the Gaussian 09 program. The
optimized structures of Mito-CP and Mito-Q are provided in
Figure S10. The highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO) of Mito-
CP and Mito-Q are given in Figure S11. For the cleavable
product Mito-Q, the π electrons resides mainly on the N, N-
dimethylamino group in the highest occupied molecular
orbital (HOMO), whereas the LUMO is mostly located on the
quinoline group with a bigger energy gap (5.13 eV) than that
of Mito-CP (3.31 eV), which is in good agreement with the
apparent blueshift in absorption spectra of Mito-Q compared
with that of Mito-CP. All these results validated our design
that the reaction of Mito-CP with Cys causes the cleavage of
acrylate and subsequent 1,6-elimination of p-hydroxybenzyl
moiety to yield Mito-Q.
Detection of Cys with Mito-CP in live cells. Standard 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay using PC12 cells was performed to assess the
cytotoxicity of Mito-CP. A >90% survival rate of cells was
maintained even with the Mito-CP concentration as high as 30
µM (Figure S12). To evaluate the performance of Mito-CP in
cultured cells, we imaged Cys with Mito-CP in PC12 cells.
When the PC12 cells were incubated with Mito-CP, a red
fluorescence signal was observed due to the presence of
endogenous Cys in cells (Figure 2a). The fluorescence
intensity increased gradually with the extension of incubation
time and reached the maximum at 30 min (Figure S13). When
the PC12 cells were first pretreated with N-ethylmaleimide
(NEM, a recognized thiol eliminator29) and then with Mito-CP,
nearly no fluorescence signal was detected (Figure 2b),
indicating that the endogenous Cys was exhausted by NEM.
When excess exogenous Cys was introduced into cells after
treating with NEM, we again observed strong intracellular
To confirm the reaction mechanism, the reaction mixture of
Mito-CP and Cys was analyzed by HRMS. After reacting with
Cys, the peak at m/z = 517.1933 declined and a new peak at
m/z = 357.1422 corresponding to Mito-Q appeared. This
verified that probe Mito-CP can be cleaved by Cys to produce
Mito-Q. (Figure S9). In order to further evaluate the optical
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fluorescence (Figure 2c). Conversely, the addition of external
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as compared to 2a). The intracellular fluorescence intensities
of the above seven groups of cells were calculated and
presented in Figure 2h.
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Hcy or GSH to the NEM-treated PC12 cells did not cause a
detectable fluorescence signal (Figure 2d, e). These imaging
phenomena are consistent with the results in solutions, further
verifying that the probe Mito-CP can be used to selectively
detect Cys in various environments. Dithiothreitol (DTT) is a
known stimulus to produce Cys in cells30. When the NEM-
treated PC12 cells were incubated with DTT, elevated
fluorescence was detected (Figure 2f), further confirming the
ability of Mito-CP to track the endogenous Cys. Moreover, we
administrated the cells with external H2O2 to imitate
intracellular oxidative stress. In this case, much weaker
intracellular fluorescence was observed in the cells (Figure 2g,
To identify the intracellular localization of Mito-CP, we
performed fluorescence colocalization experiments employing
organelle trackers (Mito-, ER-, and Lyso-Tracker Green). The
fluorescence of Mito-CP overlapped well with that of Mito-
Tracker (with a Pearson’s coefficient (PC) of 0.897) but only
partially overlapped with Lyso-Tracker (PC 0.709) or ER-
Tracker (PC 0.724), demonstrating that Mito-CP distributes
mainly in mitochondria (Figure S14). This can be explained by
the charge attraction between Mito-CP with a positive charge
and cellular mitochondrial membrane with negative potential.
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Figure 2. Fluorescence images of PC12 cells, pictures below are corresponding bright-field photos. (a) PC12 cells incubated with
Mito-CP (10 μM) for 30 min. (b) PC12 cells pretreated with NEM (1.0 mM) for 30 min and then incubated with Mito-CP (10 μM)
for 30 min. (c,d,e) PC12 cells pretreated with NEM (1.0 mM) for 30 min and then incubated with Cys, Hcy, and GSH (100 μM) for
30 min, respectively, and finally incubated with Mito-CP (10 μM) for 30 min. (f) PC12 cells pretreated with NEM (1.0 mM) for 30
min and then incubated with DTT (1.0 mM) and then incubated with Mito-CP (10 μΜ) for 30 min. (g) PC12 cells pretreated with
H2O2 (200 μM) for 30 min and then incubated with Mito-CP (10 μΜ) for 30 min. (h) Relative pixel intensity of the corresponding
fluorescence images in a-g. Scale bar = 10 μm.
Fluorescence Imaging of Cys in Living Zebrafish under
Oxidative Stress. With the promising imaging performance
in live cells in hand, we were interested in investigating the
Cys fluctuation under oxidative stress status using zebrafish as
a model. Oxidative stress refers to the cellular status with
enhanced production of intracellular reactive oxygen species
and a decrease of Cys concentration level, which impairs the
function of the cellular antioxidant defense system.31,32 The
oxidative stress status of zebrafish was mediated by
lipopolysaccharide (LPS) in our experiments, and then the
zebrafish were imaged at a set of time points from 30 to 180
min. As shown in Figure 3, clear fluorescence was observed
for the control group without LPS administration, but a
decrease of fluorescence was observed for the LPS (100
μg/mL) stimulated zebrafish that was co-incubated with Mito-
CP. The mean fluorescence intensity of zebrafish declined
with time, which was in line with the reduced concentration of
Cys in oxidative stress status. Therefore, the probe Mito-CP
was demonstrated to be able to image fluctuation of
endogenous Cys in living animals.
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Figure 3. Fluorescence images of probe Mito-CP responding to Cys in living 5-day-old zebrafish under oxidative stress status by
confocal fluorescence imaging. (a) Zebrafish were incubated with Mito-CP (10 μM, 60 min), and then imaged. (b−g) Zebrafish
were mediated by lipopolysaccharide (LPS, 100 μg/mL) for 30 (b), 60 (c), 90 (d), 120 (e), 150 (f), or 180 (g) min, and subsequently
incubated with Mito-CP (10 μM) for 60 min, and then imaged. (h) Mean fluorescence intensity of zebrafish shown in (a-g).
Fluorescence imaging of Cys in the brains of mice. Since
the emission of the probe is in the NIR region, it causes very
slight photo-damage to biological samples. In particular, it
could penetrate deeper tissues with weak background
fluorescence interference. These merits make Mito-CP well
suited for in vivo imaging applications. Therefore, we
exploited in vivo fluorescence imaging of Cys with the probe
in mice. In order to possibly cross the BBB, the lipophilicity
index log P of a molecule probe normally needs to be within
the range of 1.0-3.0, with the molecular weight (MW) < 600
Da. The log P value of Mito-CP was calculated as 1.98,
fulfilling the criterion of lipophilicity. At the same time, we
avoided constructing too large a conjugated system for the red
shift of the emission wavelength, so that Mito-CP also
possessed an appropriate MW (517 Da). The BBB
penetrability of Mito-CP was then experimentally confirmed
by ex vivo fluorescence imaging. After intravenous (i.v.)
injection of the probe, the fluorescence at 700 nm of various
organs were measured and compared. The images of both the
entire organs (Figure S15) and their sliced tissues (Figure S16)
indicated Mito-CP could reach brains through i.v. injection
and achieve ideal images. Besides, the major organs of mice
from the control and experiment groups were collected for
histological analysis. As shown in Figure S17, there was
negligible organ damage (no necrosis, edema, inflammatory
infiltration, or hyperplasia) in the sections of the six organs,
demonstrating that Mito-CP has good biosafety. To investigate
whether Mito-CP can be used for mapping the dynamic
changes of Cys in vivo, a group of five-week-old BALB/c
nude mice were studied with intraperitoneal (i.p.) injection of
different agents to induce the changes of endogenous Cys.
Images were collected at different time points after
intravenous (i.v.) injection of Mito-CP (Figure 4). As
displayed in Figure 4a and 4b, a bright fluorescence signal was
detected in the control group, which indicates Mito-CP can
effectively penetrate the BBB of healthy mice and react with
the intrinsic Cys in the brain. Higher fluorescence intensity
was observed in the brain region of mice injected with Cys
than that in the control sample. In contrast, when the BALB/c
nude mice were pretreatment with H2O2 or NEM, the NIR
fluorescence signals in brains were lower than that in the
control group. At all the time points tested, i.e., 5, 15, 30, 45,
and 60 min, the fluorescence intensity of four sample groups
all displayed the same tendency. When fluorescence imaging
was carried out on the dissected brains of the four groups, the
same result was obtained (Figure 4c). These findings verified
that Mito-CP was capable of detecting Cys levels in the brains
of living body.
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(c)
Low
Figure 4. (a) Mapping Cys fluxes in live mice with Mito-CP. 5-weeks-old BALB/c nude mice were performed with intraperitoneal
(i.p.) injection of different agents. Images were recorded after i.v. injection with Mito-CP at 5, 15, 30, 45, and 60 min. (b)
Quantification of fluorescence intensity of images in (a). (c) The Fluorescence images of dissected brain of the four mice.
Revealing Cys variation in the brains of Epileptic mice.
With the sensitive and specific response of Mito-CP towards
Cys established both in vitro and in vivo, we were then
encouraged to explore the participation of Cys in the seizure
and treatment of epilepsy by drugs in mice. Epilepsy was
induced on a mouse model by intraperitoneal injection of
pentylenetetrazole (PTZ) into the nude mice (Figure S18).
PTZ is a GABAA antagonist commonly used as a convulsant
agent, which slowly results in the development of a kindled
model of epilepsy when administered repeatedly in a sub-
convulsive dose for a specific period of time.33 All mice
samples were intravenously injected with Mito-CP, and
fluorescence imaging was performed at different time post
injection. As shown in Figure 5a and 5b, the brains of mice
with epilepsy showed evident decreased fluorescence intensity
compared to those of normal mice as the control, which
demonstrates that the probe could visualize the down-
regulation of Cys in the epileptic brains. However, when the
epileptic mice were treated with the antiepileptic drug
curcumin34-35, the fluorescence in the brain region was restored
and close to that of normal mice. The dissected brain of each
mouse was also imaged, which showed the same result as that
in vivo (Figure 5c). Overall, these results provided direct
evidence for the negative correlation between Cys levels and
epilepsy behaviors. Furthermore, the brain tissue of each
group was analyzed by hematoxylin and eosin (H&E) staining
(Figure 5d). The normal control group showed a clear
hierarchical structure, neatly arranged nerve cells, intact cell
membranes and uniform cytoplasmic staining. On the contrary,
severe neuronal death, including neuronal loss and disordered
arrangement in hippocampal regions particularly in the CA1
and CA3 regions, could be obviously observed after the
administration of PTZ. The epileptic and curcumin-treated
group presented a similar H&E staining result with the control.
These results were in good agreement with the observation of
in vivo fluorescence imaging.
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Figure 5. (a) In vivo NIR fluorescence imaging with Mito-CP. From top to bottom, images of healthy mice, epileptic mice and
curcumin-treated epileptic mice at 5, 15, 30, 45, and 60 min after intravenous injection (i.v.) of Mito-CP. (b) Quantification of
images in (a). (c) The Fluorescence images of separated brain from the mice. (d) H&E staining for evaluating neuronal damage in
the hippocampal region after PTZ administration.
CONCLUSIONS
AUTHOR INFORMATION
Corresponding Author
In summary, we created a novel NIR emissive fluorescent
probe named Mito-CP for the detection of Cys, which
possessed high sensitivity and specificity toward Cys. Mito-
CP also featured good mitochondria-targeting and BBB
penetrating abilities. Using this probe, we achieved in-situ
visualization of the Cys variations in live cells and zebrafish
with oxidative stress under external stimulation. The NIR
emission and large Stokes shift of Mito-CP enabled it to
monitor Cys in the brains of live mice. Ultimately, we were
able to explore for the first time the participation of Cys
during the seizure and treatment of epilepsy on mouse model.
Our results provided fresh insight into the relationship
between mitochondrial Cys level, an indicator of oxidative
stress, and the occurrence, development and treatment of
epilepsy.
*E-mail: zhenli@whu.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was financially supported by the National Natural
Science Foundation of China (No. 21575109, 21625503).
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
1H NMR, 13C NMR and MS spectra, additional spectroscopic data,
cell cytotoxicity and supplemental fluorescence images.
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