This document describes experiments investigating the effects of clomipramine in models of multiple sclerosis. Clomipramine showed neuroprotective effects against iron-mediated toxicity in neurons in culture. It also demonstrated antioxidant properties and reduced T-lymphocyte and B-lymphocyte proliferation. In mouse models of EAE, clomipramine decreased disease burden and inflammation when administered from disease onset. Further experiments explored the effects of clomipramine in various EAE models induced with different antigens. Overall, the results suggest clomipramine has therapeutic potential for reducing inflammation and neurodegeneration in multiple sclerosis.

1. Published on :- 19th December 2017
Presented by :- Jalormi Parekh
M.Sc. MBT Previous

2. What is Multiple Sclerosis?
• Multiple sclerosis is an inflammatory condition of the CNS
leading to damage of the myelin sheath.
• Immune system attacks the protective myelin sheath and
causes communication problem between brain and rest of the
body.

3. TYPES OF MULTIPLE SCLEROSIS

4. SYMPTOMS

5. HOW DOES INFLAMMATORY DEMYELINATION
AND NEURODEGENERATION OCCUR ?
BLOOD BBB CNS

6. PREVIOUS STUDIES
Results of two Phase 3 trials of ocrelizumab
in primary progressive. Ocrelizumab has since been
approved for use in primary progressive MS .Montalban et al(2017)
The central role of mitochondria in axonal degeneration and
demyelination in Multiple Sclerosis. Campbell, G. R., Worrall, J. T. &
Mahad, D. J. (2014)
Clomipramine suppresses clinical signs and T and B cell response
to myelin proteins in experimental autoimmune neuritis in Lewis
rats. Zhu, J. et al. (2016)
Iron causes neurodegeneration by getting accumulated in the
external region of neuron and taken up by microglia and hence
causes demyelination . Hametner, S et al (2013)

7. AIM
To check the effect of Clomiramine on T-Cell
Lymphocyte and B-Cell Lymphocyte in the
Experimental Autoimmune Enchephalomyelitis (EAE)
model of mice.

8. EXPERIMENT 1:-TO CHECK THE EFFECT OF
GENERIC DRUGS AGAINST IRON MEDIATED
AND ROTENONE NEUROTOXICITY IN
NEURONS.

9. PROTOCOL:-
Neurons labelled with
MAP-2 pre-incubated
with drugs(10 µm) for
1 hour.
Addition of FeSO4
(25µM/50µM)
Generic drugs tested against iron toxicity on human
neurons in culture.

10. PROTOCOL:-
Condition:-37˚C and
5% CO2
After addition of
FeSO4 to neurons
Add Hoechst
33342(1:2 diluted in
AIM-V medium)+ PI
(1:20 diluted in AIM-V
medium)
Live Cell Imaging of neurons

11. LIVE CELL IMAGING

12. • Rotenone induces
neurotoxicity by
binding to the
complex 1 of the
respiratory chain and
preventing electron
transfer to
ubiquinone.
Effect against rotenone neurotoxicity

13. The scale bars depict 100 μm
Live cell imaging was carried out further and neurons
were detected using anti-MAP-2 antibody.
It can be concluded that Clomipramine with other types of drugs
showed significant effect against iron mediated neurotoxicity and
rotenone neurotoxicity.

14. EXPERIMENT 2:- TO CHECK THE
HYDROXYL RADICAL SCAVENGING
CAPACITY OF THE DRUGS

15. OH-
ANTIOXIDANT
FLUORESCENCE DECREASES RAPIDLY
FLUORESCENCE DECREASES SLOWLY
Hydroxyl Radical Antioxidant Capacity Assay
•Investigates the prevention of hydroxyl radical mediated oxidation of fluorescein in
comparison to the strong antioxidant gallic acid.
Progressive loss of fluorescence:
Cobalt-driven Fenton-like reaction oxidizes
fluorescein

16. Hydroxyl Radical Antioxidant Capacity Assay

17. It can be concluded that clomipramine (HORAC-GAE= 2.1) along with
certain other drugs showed significant antioxidative property
HORAC ASSAY

18. EXPERIMENT 3:- TO TEST THE EFFECT OF
DRUGS ON T-CELL PROLIFERATION.

19. Clomipramine attenuated the activation of T-Cell Lymphocyte
ACTIVITY OF DRUGS AGAINST T-CELL PROLIFERATION

20. EXPERIMENT 4:- FOCUS ON
CLOMIPRAMINE IN VIVO

21. FOLLOWING WERE THE REASONS TO TAKE
CLOMIPRAMINE IN FURTHER STUDIES
Strong effects against iron mediated neurotoxicity (mean %
anti-MAP-2 positive cells normalized to control of 107.3%) and
neuroprotection against iron toxicity from 100nM
Has antioxidative properties (HORAC-GAE 2.1)
Reduced T-Lymphocyte proliferation (68.2%) in a concentration
dependent manner from 5µM
BCR/anti-CD-40L/IL-4 activation of B-cells increased
proliferation and production of TNF-α and these were reduced
in a concentration dependent manner by clomipramine from
2µM

23. Clomipramine was thus used for phase 2 trails.

24. EXPERIMENT 5:-TO INVESTGATE
CLOMIPRAMINE IN ACUTE
EAE(Experimental Autoimmune
Encephalomyelitis) MODEL OF MICE

25. WHAT IS EAE (Experimental Autoimmune
Encephalomyelitis)?
• Complex condition–interaction between immunopathological
and neuropathological mechanism leads to key feature of
Multiple Sclerosis-inflammation, demyelination, axonal loss.
• Major difference between MS and EAE:- EAE requires
external immunization step to develop which occurs through
use of adjuvant and contains bacterial component highly
capable of activating the innate immune system.
• Demyelination in EAE:- Injection of brain extracts such as
MOG, SCH, CNS peptides emulsified in Freund adjuvant. Has
inactivated and dried mycobacteria.
• Limitation to EAE:- Oxidative Injury and B-cell proliferation.

26. Therapy of clomipramine from day 5 of induction of MOG-
EAE in C57BL/6 mice.

27. Therapy of clomipramine from the day of onset of disease and checking the
weight of the mice.

28. Transcript level of pro-inflammatory factor

29. Investigation of serum levels and spinal cord of clomipramine
and its active metabolite, desmethylclomipramine (DMCL)

30. Check the parenchymal inflammation (H&E Staining)
Check the microglial activation (Iba1 Staining)
Axonal damage (Bielschowsky Staining)
It can be concluded that when Clomipramine was give to the MOG-
immunized acute-EAE model of mice, it decreased the disease burden
and even reduced the inflammation .

31. EXPERIMENT 6:-TO INVESTGATE
CLOMIPRAMINE IN CHRONIC
EAE(Experimental Autoimmune
Encephalomyelitis) MODEL OF
MICE

32. Effect of clomipramine from first relapse
No significant difference was seen in between the vehicle and the
Clomipramine treated mice during the remission phase.

33. EXPERIMENT 7:-TO INVESTGATE
CLOMIPRAMINE IN MOG-
IMMUNIZED C57BL/6 MODEL OF
MICE

34. Treatment of mice from the first onset of clinical
signs
The disease burden was less as compared to that of vehicle during the
first and second relapsing phase and no significant difference is seen
during the remission phase post immunization.

35. EXPERIMENT 8 :-TO INVESTGATE
CLOMIPRAMINE IN SCH-
IMMUNIZED BIOZZI ABH MODEL
OF MICE

36. Secondary model to study multiple sclerosis.
Immunized with Spinal Cord Homogenate (SCH)
Clomipramine treatment from onset of disease.
The Biozzi ABH mice model with SCH immunization and Clomipramine
treatment was showing less disease burden when compared to
vehicle.

37. SUMMARY

38. MULTIPLE SCLEROSIS :
INFLAMMATION /
INFLAMMATORY EFFECT
DRUGS
OXIDATIVE STRESS
IRON MEDIATED
NEUROTOXICITY

39. MULTIPLE SCLEROSIS :
INFLAMMATION /
INFLAMMATORY EFFECT
OXIDATIVE STRESS
IRON MEDIATED
NEUROTOXICITY
DRUGS

40. EAE
MOG/SCH
IMMUNIZATION
PBS
DISEASE BURDEN AT
PEAK
DECREASE IN THE
DISEASE BURDEN

43. BACK UP

45. Cell culture and treatment of human neurons.
• Human neurons were isolated from brain tissues of aborted 15–20-week-old fetuses.
• Neurons were isolated as follows in brief: brain specimens were washed in phosphate
buffered saline (PBS) to remove blood, followed by removal of meninges.
• Tissue was mechanically dissected, followed by digestion in DNase (6–8ml of 1 mg/ml;
Roche), 4ml 2.5% trypsin and 40 ml PBS (37 °C, 25 min).
• Thereafter, the digestion was stopped by addition of 4ml fetal calf serum. The solution
was filtered through a 132 μm filter and centrifuged (three times, 1200 rpm, 10 min).
• Cells were cultured in feeding medium of minimal essential medium supplemented
with 10% fetal bovine serum (FBS), 1 μM sodium pyruvate, 10 μM glutamine, 1× non-
essential amino acids, 0.1% dextrose, and 1% penicillin/streptomycin .
• The initial isolates of mixed CNS cell types were plated in poly-Lornithine-coated (10
μg/ml) T75 flasks and cultured for at least two cycles44 in medium containing 25 μM
cytosine arabinoside to inhibit astrocyte proliferation and to deplete this major
contaminating cell type.
• For experiments, the neuron-enriched cultures were re-trypsinized and cells were
plated in poly-L-ornithine pre-coated 96-well plates at a density of 100,000 cells/ well in
100 μl of the complete medium supplemented with cytosine arabinoside. Medium was
changed to AIM V® Serum Free Medium (Invitrogen) after 24 h.
• After a period of 1 h, respective drugs were added in a concentration of 10 μM,
followed by application of FeSO4 or rotenone after 1 h.

46. PROLIFERATION OF T-CELL LYMPHOCYTES

47. Harvest Spleens from female C57BL/6 mice
mechanical dissociation of spleen
pass cell through a 70 μm cell strainer and separated by Ficoll gradient (1800
RPM, 30 min). Treat wells drugs 10 μM.
plate (2.5 × 105 cells in 100 μl/well) in anti-CD3 antibody-coated 96-
well plates (1000 ng/ml plate-bound anti-CD3 and 1000 ng/ml anti-CD28
suspended in media) Culture medium:- (RPMI 1640 medium, supplemented
with 10% FBS, 1 μM sodium pyruvate, 2 mM L-alanyl-L-glutamine, 1%
penicillin/streptomycin, 1% HEPES, and 0.05mM 2- mercaptoethanol).
After 48 hours add 3H-thymidine (1 μCi per well). Harvest cell after 24 h on
filter mats.

48. Activity on B-lymphocytes.
• Venous blood from healthy volunteers was obtained and peripheral
blood mononuclear cells (PBMCs) were isolated by Ficoll gradient
centrifugation (1800 RPM, 30 min).
• From PBMCs, B-cells were isolated by positive selection with CD19-
directed microbeads .
• Purity was assessed by FACS after staining for CD19 .
• Cells were plated at a concentration of 2.5 × 105 cells/well in X-
VIVO medium (Lonza) supplemented with 1%
penicillin/streptomycin and 1% Glutamax and treated with drugs for
1 h.
• Cells were then activated with 10 μg/ml IgM BCR cross-linking
antibody (XAb) (Jackson ImmunoResearch), 1 μg/ml anti-CD40L and
IL-4 20 ng/ml for 24 h as described previously48. Conditioned
media were harvested after 24 h for ELISA.
• Medium as well as respective drugs were re-added followed by
application of 3H-thymidine in a concentration of 1 μCi per well to
investigate proliferation.
• After 24 h, cells were harvested on filter mats and after drying cpm
were measured using a liquid scintillation counter.

49. EAE induced in 8–10-week old female C57BL/6 mice. Mice
injected with 50 μg of MOG35–55 in Complete Freund's
Adjuvant supplemented with 10 mg/ml Mycobacterium
tuberculosis subcutaneously on both hind flanks on day 0.
Pertussis toxin (300 ng/200) injected intraperitoneal (IP) on days
0 and 2.
Animals treated with clomipramine (25 mg/kg; 100 μl of 5
mg/ml solution) by IP injection from day 0 or day 5, from day
30 at remission and from 13 at onset of clinical signs.
( The solution of clomipramine was prepared daily in PBS.)
GENERATION OF EAE IN C57BL/6
MICE

50. GENERATION OF EAE IN BIOZZI ABH
MICE
• EAE induced in Biozzi ABH mice aged 8–10 weeks by
subcutaneous application of 150 μl emulsion in both sides of
the hind flanks.
• The emulsion contains: Stock A:- (4ml of incomplete Freund's
adjuvant mixed with 16 mg M. tuberculosis and 2mg M.
butyricum). Stock B:- (1 ml of stock A + 11.5 ml of incomplete
Freund adjuvant)
• Stock B mixed in equal volume with SCH in PBS before
injection. SCH used in a concentration of 6.6 mg/ml emulsion
each for 2 injections (days 0 and 7).

51. FLOW CYTOMETRY
FACS CELL CYCLE STUDY

52. Flow Cytometry and FACS
• FACS:- It provides a method for sorting a heterogeneous mixture of biological
cells into two or more containers, one cell at a time, based upon the specific light
scattering and fluorescent characteristics of each cell.
• The cell suspension is entrained in the center of a narrow, rapidly flowing stream of
liquid.
• The flow is arranged so that there is a large separation between cells relative to their
diameter. A vibrating mechanism causes the stream of cells to break into individual
droplets.
•The system is adjusted so that there is a low probability of more than one cell per droplet.
• Just before the stream breaks into droplets, the flow passes through a fluorescence
measuring station where the fluorescent character of interest of each cell is measured.
• An electrical charging ring is placed just at the point where the stream breaks into
droplets.
• A charge is placed on the ring based on the immediately prior fluorescence intensity
measurement, and the opposite charge is trapped on the droplet as it breaks from the
stream.
• The charged droplets then fall through an electrostatic deflection system that diverts
droplets into containers based upon their charge.

53. Cell cycle study:-
•Cell cycle analysis by DNA content measurement is a method that most frequently
employs flow cytometry to distinguish cells in different phases of the cell cycle.
• Before analysis, the cells are usually permeabilised and treated with a fluorescent dye that
stains DNA quantitatively, such as propidium iodide (PI) or 4,6-diamidino-2-phenylindole
(DAPI).
•The fluorescence intensity of the stained cells correlates with the amount of DNA they
contain.
• As the DNA content doubles during the S phase, the DNA content (and thereby intensity of
fluorescence) of cells in the G0 phase and G1 phase (before S), in the S phase, and in
the G2 phase and M phase (after S) identifies the cell cycle phase position in the major
phases (G0/G1 versus S versus G2/M phase) of the cell cycle.
•The cellular DNA content of individual cells is often plotted as their frequency histogram to
provide information about relative frequency (percentage) of cells in the major phases of
the cell cycle.

54. RT-PCR
•Quantitative reverse transcription PCR (RT-qPCR) is used when the starting material is
RNA.
• RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase from
total RNA or messenger RNA (mRNA).
•The cDNA is then used as the template for the qPCR reaction. RT-qPCR is used in a variety
of applications including gene expression analysis, RNAi validation, microarray validation,
pathogen detection, genetic testing, and disease research.

55. RT-PCR
• Lumbar spinal cords were harvested, snap frozen in liquid nitrogen
and stored in −80 °C.
• Samples were homogenized in 1 ml Trizol followed by the Addition
of 200 μl chloroform.
• The suspension was shaken, centrifuged (11,500 RPM for 15 min at
4 °C) and the RNA-containing upper phase was transferred into a
new tube and precipitated with equal amounts of 70% ethanol.
• RNA was extracted.
• RNA concentrations were measured.
• cDNA preparation was performed with 1 μg of RNA.
• Real-time PCR was performed and primers for Gapdh(Qiagen) as
housekeeping gene, Ifn-γ (Qiagen, QT01038821), Tnfa (Qiagen,
QT00104006), Il-17 (SABiosciences, PPM03023A-200), and Ccl2
(Qiagen, QT00167832). Relative

56. LC-MS
• Liquid chromatography–mass spectrometry (LC-MS) is an analytical
chemistry technique that combines the physical separation
capabilities of liquid chromatography (or HPLC) with the mass
analysis capabilities of mass spectrometry(MS).
• Coupled chromatography - MS systems are popular in chemical
analysis because the individual capabilities of each technique are
enhanced synergistically.
• While liquid chromatography separates mixtures with multiple
components, mass spectrometry provides structural identity of the
individual components with high molecular specificity and
detection sensitivity.
• This tandem technique can be used to analyze biochemical, organic,
and inorganic compounds commonly found in complex samples of
environmental and biological origin.
• Therefore, LC-MS may be applied in a wide range of sectors
including biotechnology, environment monitoring, food processing,
and pharmaceutical, agrochemical, and cosmetic industries.