Unambiguous Detection of Elevated Levels of Hypochlorous Acid in Double Transgenic AD Mouse Brain

Article abstract of DOI:10.1021/acschemneuro.9b00554

Alzheimer's disease (AD) is one of the most prevalent forms of dementia. The current diagnosis methods based on the behavior and cognitive decline or imaging of core biomarkers, namely, amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs), in the brain offer poor to moderate success. Detection and imaging of biomarkers that cause additional traits of pathophysiological aberrations in the brain are invaluable to monitor early disease onset and progression of AD pathology. The pathological hallmark of AD is associated with generation of excessive reactive oxygen species (ROS) in the brain, which aggravate oxidative stress and inflammation. ROS production involves elevated levels of hypochlorous acid (HOCl) and can serve as one of the potential biomarkers for the diagnosis of AD. We report the design, synthesis, and characterization of switchable coumarin-morpholine (CM) conjugates as off-on fluorescence probes for the specific detection of HOCl produced and proximally localized with amyloid plaques. The nonfluorescent thioamide probe CM2 undergoes regioselective transformation to fluorescent amide probe CM1 in the presence of HOCl (～90-fold fluorescence enhancement and 0.32 quantum yield) with high selectivity and sensitivity (detection limit: 0.17 μM). The excellent cellular uptake and blood-brain barrier (BBB) crossing ability of CM2 allowed unambiguous and differential detection, imaging, and quantification of HOCl in cellular milieu and in the wild type (WT) and AD mouse brains. This study demonstrates the elevated level of HOCl in the AD mouse brain and the potential to expand the repertoire of biomarkers for the diagnosis of AD.

Full text of DOI:10.1021/acschemneuro.9b00554

Research Article
pubs.acs.org/chemneuro
Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Unambiguous Detection of Elevated Levels of Hypochlorous Acid in
Double Transgenic AD Mouse Brain
Sourav Samanta and Thimmaiah Govindaraju*
Bioorganic Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O.,
Bengaluru 560064, Karnataka, India
S
* Supporting Information
ABSTRACT: Alzheimer’s disease (AD) is one of the most prevalent forms of
dementia. The current diagnosis methods based on the behavior and cognitive decline
or imaging of core biomarkers, namely, amyloid-β (Aβ) plaques and neurofibrillary
tangles (NFTs), in the brain offer poor to moderate success. Detection and imaging of
biomarkers that cause additional traits of pathophysiological aberrations in the brain are
invaluable to monitor early disease onset and progression of AD pathology. The
pathological hallmark of AD is associated with generation of excessive reactive oxygen
species (ROS) in the brain, which aggravate oxidative stress and inflammation. ROS
production involves elevated levels of hypochlorous acid (HOCl) and can serve as one
of the potential biomarkers for the diagnosis of AD. We report the design, synthesis,
and characterization of switchable coumarin-morpholine (CM) conjugates as off-on
fluorescence probes for the specific detection of HOCl produced and proximally
localized with amyloid plaques. The nonfluorescent thioamide probe CM2 undergoes
regioselective transformation to fluorescent amide probe CM1 in the presence of HOCl (∼90-fold fluorescence enhancement
and 0.32 quantum yield) with high selectivity and sensitivity (detection limit: 0.17 μM). The excellent cellular uptake and
blood-brain barrier (BBB) crossing ability of CM2 allowed unambiguous and differential detection, imaging, and quantification
of HOCl in cellular milieu and in the wild type (WT) and AD mouse brains. This study demonstrates the elevated level of
HOCl in the AD mouse brain and the potential to expand the repertoire of biomarkers for the diagnosis of AD.
KEYWORDS: Alzheimer’s disease, diagnosis, HOCl, regioselective thioamide to amide transformation, brain imaging
biomarkers list open ended to add newly validated biomarkers
that are reliable and quantifiable. This encourages researchers
to validate newer biomarkers to improve the accuracy of
diagnosis, as core biomarkers are not completely successful in
delivering an accurate diagnostic platform for multifaceted
AD.15−21 The weak inorganic acid HOCl (pKa= 7.5) plays a
detrimental role in the process leading to oxidative stress and
neuroinflammation and hence is a potential candidate as AD
biomarker.22 The redox active transition metal ions (CuII and
FeIII) chelate with Aβ and stabilize highly toxic metal-
dependent Aβ-aggregation species which create severe
imbalance in the physiological redox homeostasis in the AD
brain.6,21,23 The bound metal ions (CuII or FeIII) undergo two
electron reduction in the presence of the host Aβ peptide or
other biological reducing agents to generate excessive hydro-
gen peroxide (H2O2) from molecular oxygen.6,24 In the
subsequent transformation, myeloperoxidase enzyme in the
presence of chloride ions coverts H2O2 to HOCl.25 HOCl is a
strong oxidizing agent and helps in an array of essential
biological processes such as fighting against external pathogens,
regulation of growth factors, stimulation of healing, modulation
INTRODUCTION
■
Alzheimer’s disease (AD) is a major chronic and progressive
neurological disorder constituting 70−80% of all dementia
cases.1 The disease pathology is characterized by the presence
of toxic Aβ plaques and neurofibrillary tangles (NFTs) in the
brain.2−6 The population affected by AD has been increased by
more than 71% (as of 2013) while that of major diseases such
as AIDS, cancer, and heart diseases shows decline owing to
availability of reliable treatments.6,7 The situation is alarmingly
frightening, as more than 50 million people worldwide will be
affected by dementia by 2050, of which 70−80% accounts for
AD.1,7 Currently, AD is detected only in the advanced stage at
which there are no treatment options available.8,9 AD is
characterized by multifaceted toxicity that involves multiple
biomarkers, and all these biomarkers must be effectively
addressed individually as well as collectively through reliable
analytical tools.10−13 Therefore, the development of analytical
tools for biomarkers that have a direct or indirect role in AD
pathogenesis demands immediate attention to aid multiplexing
and multimodal analysis.6 The National Institute on Aging and
Alzheimer’s Association (NIA-AA) Research Framework
report (2018) recommended the use of definite biomarkers
instead of cognitive tests to diagnose AD and proposed Aβ,
tau, and related neurodegeneration as core biomarkers.14 The
NIA-AA Research Framework has consciously left the
Received: October 12, 2019
Accepted: December 2, 2019
Published: December 2, 2019
© XXXX American Chemical Society
A
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Figure 1. (A) Synthesis of CM probes. (a) Diethylmalonate, piperidine, EtOH; (b) conc. HCl; (c) morpholine, HBTU, HOBt, DIPEA, DMF; (d)
Lawesson’s reagents, THF. (B) ORTEP diagram of single crystal X-ray diffraction of CM2. (C) Fluorescence emission spectra of CM2 in the
absence (broken line) and presence (thick line) of OCl−, and corresponding spectra of CM1 are shown in the inset. (D) HPLC analysis of CM1,
CM2, and HOCl treated CM2, and characteristic retention time (tR) is indicated. (E) Schematic diagram show the AD brain with Aβ deposition,
Aβ-metal complex catalyzed production of elevated levels of HOCl and its in vivo detection using BBB permeable CM2. NFI: Normalized
fluorescence intensity.
of inflammation, and posttranslational modifications
(PTMs).26 Several HOCl sensors have been reported in the
literature for HOCl sensing,27−33 while the detection or
imaging of HOCl and its relation to Aβ (production and
localization) under in vitro and in vivo AD-like conditions is
yet to be established to validate HOCl as a potential
biomarker. A simple, accurate, and reliable method for the
detection and imaging of HOCl in the AD brain is useful for
the effective diagnosis of AD pathology. In this Article, we
report a blood-brain barrier (BBB) permeable thioamide probe
CM2 for highly specific and sensitive detection, imaging, and
quantification of HOCl under in vitro and in vivo AD-like
conditions to validate it as one of the prospective biomarkers
for the diagnosis of AD (Figure 1).
acid I2 was condensed with morpholine using HBTU [(2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate] and HOBt (1-hydroxybenzotriazole) in dimethylforma-
mide (DMF) to obtain CM1 (7-(diethylamino)-3-(morpho-
line-4-carbonyl)-2H-chromen-2-one). The amide carbonyl
oxygen atom in CM1 was substituted with sulfur atom using
Lawesson’s reagent to obtain CM2. The integrity of all the
intermediates and probes (CM1 and CM2) was confirmed by
NMR, HPLC, and high-resolution mass spectrometry
(HRMS) techniques. The structure of the probe CM2 was
confirmed by single crystal X-ray diffraction analysis, which
revealed planar and chair conformations of coumarin and
morpholine units, respectively (Figure 1B). The single crystal
XRD structure confirmed the regioselective amide (CM1) to
thioamide (CM2) conversion without affecting the lactone
carbonyl of the coumarin moiety.
Regioselective Oxidation of CM2 and HOCl Detec-
tion. As anticipated, CM2 rapidly reacted with HOCl in
solution and showed bright green fluorescence (λex = 417 nm,
λem = 485 nm), while CM1 did not show any significant
changes and remained unresponsive under similar conditions
(Figure 1C, inset). These initial findings validated our
chemical design (Supporting Information, Figure S1) and
indicated the probable transformation of nonfluorescent
thioamide (CM2) to fluorescent amide (CM1). The OCl−
oxidize the sulfur atom of the thiocarbonyl group of CM2 and
transformed into a good leaving group which is subsequently
removed by the hydrolysis to generate the carbonyl group as in
fluorescent CM1. The oxidative transformation of CM2 to
CM1 was thoroughly characterized by HPLC and HRMS
RESULTS AND DISCUSSION
■
Design and Synthesis of CM Probes. The design
strategy of CM probes was to conjugate coumarin and
morpholine units through amide (CM1) and thioamide
(CM2) linkages (Figure 1A). We anticipated that the
thioamide bond (fluorescence quencher) makes CM2 non-
fluorescent and upon transformation to CM1 with an amide
bond in the presence of HOCl restores the fluorescence of the
coumarin moiety.16,34−38 The synthetic route for the
preparation of CM1 and CM2 is shown in Figure 1A. 4-
(Diethylamino)-2-hydroxybenzaldehyde was condensed with
diethylmalonate using piperidine as a base under reflux
conditions to obtain intermediate I1. The intermediate I1
was hydrolyzed in acidic conditions to obtain 7-(diethylami-
no)-2-oxo-2H-chromene-3-carboxylic acid (I2). The carboxylic
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analysis (Figure S2). The HPLC chromatograms monitored at
420 nm showed the retention time (tR) of 5.38 and 6.65 min
for CM1 and CM2, respectively. The dichloromethane extract
of HOCl treated CM2 sample under similar conditions showed
tR = 5.38 min which confirmed the formation of CM1 (Figure
1D). HRMS analysis of the CM2 sample treated with HOCl
revealed the absence of a mass peak at 347.1434 [CM2 + H+]
and showed new mass peaks at 331.1641 and 353.1455
corresponding to [CM1 + H+] and [CM1 + Na+], respectively
(Figure S2). These results confirmed the regioselective
oxidation of CM2 at the thiocarbonyl in the presence of
HOCl to generate green fluorescent amide CM1, and the
suitability of this transformation for the detection and imaging
of HOCl (Figure 1E).
Specific Detection of HOCl over Other ROS. The
selectivity of the probe CM2 was assessed against various
different ROS including HO•, H2O2, NO2 , NO, O2, NO3 ,
tBuOOH, and OCl− in phosphate buffer saline (PBS)
containing ethanol (1:1) solution. Strong green emission was
observed in the presence of HOCl, while other ROS did not
show significant fluorescence under similar condition (Figure
2A). CM2 was evaluated in the presence of different cations
and anions to further ascertain its selectivity to HOCl. The
data confirmed the selectivity of CM2 toward OCl− over other
biological species tested to generate fluorescent CM1 (Figures
S3 and S4). Besides, we evaluated the response of CM2 in the
presence of freshly prepared monomers, oligomers, and fibrils
of Aβ42 under in vitro conditions. The fluorescence response
monitored at 485 nm did not show significant changes
compared to the strong signal from the positive control with
exogenously added HOCl (Figure S5). This study demon-
strated that the probe has no interference from the Aβ42 or
peptide species. However, in cellulo study shows a strong
response of the probe in the presence of the Aβ42+CuII
complex, which is attributed to the redox process and in situ
generation of excessive HOCl (vide infra). The hypsochromic
shift (15 nm) and hypochromic effect of absorbance maxima at
417 nm in the presence of NaOCl resulted in a colorimetric
change from yellowish to colorless solution (Figure S6). The
fluorescence emission of CM2 enhanced gradually (up to 90-
fold) as a function of NaOCl concentration and reached
saturation at 1 mM (Figure 2B). The data showed that a sub-
micromolar concentration of OCl− can be detected using CM2
with the detection limit of 0.17 μM (Figure S7). The presence
of the sulfur atom (thioamide) acts as an intramolecular
fluorescence quencher of the coumarin moiety in CM2
(quantum yield, Φ = null). The oxidative transformation of
thioamide to generate CM1 in the presence of OCl− restores
the bright green fluorescence (Φ = 0.32). Interestingly, CM2
selectively detects HOCl over a wide pH range from 3.0 to
12.0, confirming the possible utility for in vitro and in vivo
detection and imaging of HOCl in physiological conditions
without interference from other chemical species (Figure S8).
Detection of HOCl in SHSY5Y Cells Using CM2. We
evaluated the utility of CM2 to detect HOCl under complex
physiological conditions in neuronal cells (SHSY5Y). The
assessment of cytotoxicity showed that CM2 is nontoxic to
cells in the working concentration (500 nM) and exhibited cell
viability of >95% for the concentration of 10 μM, after 24 h of
incubation (Figure S9). SHSY5Y cells were cultured in
confocal (35 mm) dishes and incubated with CM2 and 4′,6-
diamidino-2-phenylindole (DAPI) for 30 min under 5% CO2
at 37 °C. The cells were washed, and images were captured
−
1
−
Figure 2. (A) Fluorescence response measured upon treatment of
−
CM2 with OCl− and various other ROS (HO•, H2O2, NO2 , NO,
−
t
1O2, NO3 , and BuOOH) in phosphate buffer saline containing
ethanol (1:1). (B) Fluorescence response recorded upon treatment of
CM2 with increasing concentration of OCl−. Insets show plot of
fluorescence intensity at 485 nm as a function of [OCl−] and visible
color change of CM2 solution on treatment with OCl− visualized
under UV light (λex = 365 nm). NFI: Normalized fluorescence
intensity.
under the fluorescence microscope in the absence and
presence of exogenous NaOCl (Figure 3). The green
fluorescence signal with the FITC channel confirmed the
cellular uptake of CM2 and its regioselective transformation to
generate fluorescent CM1. Next, SHSY5Y cells were incubated
with the Aβ42-CuII (20 μM) complex for 2 h to create in vitro
AD-like conditions and visualize the production of HOCl
under metal-dependent amyloid stress. The experimental cells
were fixed with 4% paraformaldehyde (PFA) followed by the
incubation with CM2 for 30 min, and DAPI was used to stain
the nucleus. The green fluorescence signal from the cells
revealed regioselective transformation of CM2 to CM1 and in
situ generation of HOCl as a consequence of metal-dependent
amyloid toxicity (Figure 3). The strong in cellulo fluorescence
confirmed excess production of HOCl in AD-like conditions,
which motivated us to explore the utility of CM2 for in vivo
detection and imaging of HOCl. The BBB crossing ability of
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Figure 3. Detection of HOCl in SHSY5Y cells using the CM2 probe. PBS (control), NaOCl, and Aβ42-CuII complex (2 h) treated cells were
imaged under the confocal microscope upon staining with CM2 (green) and DAPI (blue). BF, bright field; scale bar, 20 μm.
CM2 was evaluated by administering the probe in wild type
(WT) C57BL/6 mice and analyzing the brain tissue at
different time points (45 and 90 min). The mass spectrometry
analysis of the CM2 administered mouse brain samples showed
molecular peaks corresponding to [CM2]+ (346.44) and
partially transformed [CM1+2H]+ (332.17) due to the
presence of physiological HOCl (Figure S10). Further,
absorption and emission spectroscopy analysis were carried
out to reconfirm the presence of unreacted CM2 in the probe
administered mouse brain samples. The absorption spectrum
showed absorbance maxima at λ= 430 nm which revealed the
presence of unreacted CM2. However, CM2 present in the
sample reacted rapidly upon addition of exogenous OCl− and
showed a significant fluorescence enhancement at 485 nm (λex
= 417 nm). On the other hand, the PBS administered
(control) brain sample did not show any fluorescence
enhancement under similar conditions (Figure S11). These
studies have established that CM2 effectively crosses the BBB
and is a potential in vivo probe to detect and image HOCl in
the brain.
(APP) and γ-secretase (PSEN1) results in the production and
deposition of Aβ plaques in the brain. The deposition of
amyloid plaques accompanied by the accumulation of redox
active metal ions in the AD brain produce excess ROS causing
oxidative stress and neuroinflammation.6,8 To detect and image
HOCl in the mouse brain, CM2 was administered twice (12 h
interval) in AD and WT mice and the mice were sacrificed
after 6 h of a second administration to collect the brains. The
collected brains were fixed with 4% PFA and rehydrated with
30% sucrose solution to obtain the sagittal brain sections (40
μm). The brain sections were immunostained with DAPI and
amyloid fibril specific antibody (OC) followed by the
treatment with fluorescently (λex = 632 nm, λem = 647 nm)
labeled secondary antibody, and the images were captured
under the confocal microscope (Figure 4A and B). The images
obtained from two different regions of the brain viz.,
hippocampus and cortex showed green fluorescence signal
which confirmed the presence of CM1 in the CM2
administered mouse brain compare to age-matched control
(Figure 4C). The images under red fluorescence channel
displayed the deposition of amyloid plaques in the AD brain
sections. Remarkably, high resolution images showed the
localization of green fluorescence (CM1) around red
fluorescence (OC) stains in both hippocampus and cortex
regions of the AD brain (Figure 4A and B). The localization of
CM1 and OC in close proximity reveals a higher local
concentration of HOCl around Aβ aggregates and its possible
Detection, Imaging, and Quantification of HOCl in
APP/PS1Mouse Model. We proceeded with the detection
and imaging of HOCl employing CM2 in the brains of 12
month old APP/PS1 male AD mice and age matched control
(WT mice). The APP/PS1 double transgenic AD mouse
model (B6C3-Tg (APPswe, PSEN 1dE9)85Dbo/J; stock
number 004462) with mutations in amyloid precursor protein
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Figure 4. Confocal microscopy images of hippocampus (A) and cortex (B) regions of the CM2 administered WT and AD mouse brain, which are
immunostained with DAPI (blue) and amyloid fibril specific antibody (OC, red). (C) Full view of the coronal section of the AD brain; high
resolution confocal images of the specific locations of the hippocampus (broken line) and cortex (thick line) are shown in (A) and (B). (D, E)
Quantification of green-FI (HOCl levels) and red-FI (amyloid plaques) in different regions (cortex and hippocampus) of WT and AD mouse brain.
Data represent mean intensity SEM, n = 3 per group. Scale bar, 150 and 10 μm (HR); HR, high resolution; FI, fluorescence increment; Hippo,
hippocampus.
involvement in the production of excessive HOCl under AD
conditions. The CM2 administered AD mouse brain (hippo-
campus and cortex) showed significantly greater green
fluorescence (∼4- and 7-fold, respectively) than that of WT
mouse brain, which indicates higher levels of HOCl in AD
conditions (Figure 4D). Furthermore, the image of Aβ plaques
displayed an uneven amyloid burden in the cortex (∼7-fold)
and hippocampus (∼5-fold) region of AD brain. Interestingly,
the HOCl levels correlate with the amyloid burden in the
cortex (∼7-fold) as well as hippocampus (∼4-fold) regions
under AD conditions (Figure 4D and E). These proximal
localization and quantification results confirm the link between
Aβ and elevated levels of HOCl in the AD brain and qualify
HOCl as a potential biomarker for AD pathogenesis along with
amyloid plaques. These remarkable observations convincingly
demonstrate that the elevated level of HOCl in the brain is
possibly one of the definite causative factors for the oxidative
stress and neuroinflammation in AD pathogenesis. These
studies have established the selective detection, imaging, and
quantification of HOCl levels in the AD brain employing the
thioamide to amide transformation of CM probes.
vitro and in vivo detection and imaging of HOCl. The BBB
crossing ability of CM2 allowed unambiguous and differential
detection, imaging, and quantification of HOCl in the mouse
brain, which revealed elevated levels of HOCl in the AD mice
compared to age-matched control. The proximal localization of
HOCl possibly suggests its generation in the downstream
cascade of ROS production pathways of metal-amyloid
plaques. Our study established and validated that the elevated
levels of HOCl in the AD brain correlate well with the amyloid
plaques and that HOCl is a reliable biomarker to expand the
repertoire of NIA-AA biomarkers for the diagnosis of AD.
METHODS
■
In Cellulo Imaging of HOCl Under AD-like Condition.
SHSY5Y cells were treated with Aβ42+CuII complex to create AD-
like condition and excessive production of HOCl was imaged
employing CM2 probe. For this experiment, SHSY5Y cells were
cultured in 35 mm Petri dish and incubated with Aβ42+CuII (20 μM)
complex for 2 h. The cells were fixed with 4% PFA followed by
incubation with CM2 (0.5 μM) for 30 min and DAPI was used to
stain the nucleus. The cells were washed and imaged under the
confocal fluorescence microscope.
Animal Model for in Vivo Experiment. B6.Cg-Tg-
(APPswe,PSEN 1dE9)85Dbo/Mmjax mice (APP/PS1) were ob-
tained from Jackson Laboratory (stock number 004462) and
maintained and bred in the JNCASR animal facility under a 12 h
light and dark cycle. All the studies were executed according to the
guidelines of the Institutional Animal Ethics Committee (IAEC),
CONCLUSIONS
■
In conclusion, we demonstrated the design, synthesis, and
regioselective transformation of thioamide (CM2) to amide
(CM1) to serve as fluorescence off- and on-states for the in
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JNCASR. The protocol (TG001) was approved by the IAEC and the
Committee for the purpose of Control and Supervision of
Experiments on Animals (CPCSEA).
In Vivo Administration of Probe. CM2 (1 mg/kg body weight)
and vehicle (PBS) were administered into AD and WT mice (12
months, n = 3) through the nasal route. The probe (CM2)
administration was performed twice over an interval of 12 h before
sacrificing them for further studies.
Brain Collection and Immunohistochemistry. After the
administration of CM2, the mice were sacrificed through cervical
dislocation and their brains were collected. The collected brains were
fixed with 4% PFA for 48 h and rehydrated with sucrose solution
(30%). The brains were sectioned (40 μm) using a cryostat with a
−20 °C internal chamber temperature. Here, 2% BSA (bovine serum
albumin) and 1% goat serum were used to block the brain sections.
Following the blocking procedure, the sections were incubated with
primary antibodies (1:1000) for 48 h at 4 °C with gentle shaking. The
unbound primary antibody was washed, and the sections were
incubated with fluorescently (red) labeled secondary antibody
(1:300) for 4 h at room temperature in the dark with gentle shaking.
These brain sections were incubated with 4′,6-diamidino-2-phenyl-
indole (DAPI) for 10 min and taken on a glass slide for mounting
with Vectashield mounting media and then imaged using a confocal
fluorescence microscope (Olympus FV3000).
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.9b00554.
■
S
Experimental methods, characterization of probes and
intermediates, crystal structure analysis, photophysical
studies, cytotoxicity in SHSY5Y cells, mouse brain
imaging (PDF)
Crystal data for CM2 (CIF)
CheckCIF report (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: tgraju@jncasr.ac.in.
ORCID
Thimmaiah Govindaraju: 0000-0002-9423-4275
Author Contributions
S.S. and T.G. designed the project. S.S. synthesized and
undertook the photophysical, biophysical, in cellulo, and in
vivo studies under the supervision of T.G. S.S. and T.G.
prepared the manuscript.
■
Funding
We thank JNCASR, SwarnaJayanti Fellowship, DST, Govt. of
India (Grant: DST/SJF/CSA-02/2015-2016), CSIR for
student fellowship to S.S.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Prof. C. N. R. Rao FRS for constant support, Prof. A.
Anand, Dr. J. P. Clement, Dr. R. G. Prakash, and JNCASR
animal facility.
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