Professional Documents
Culture Documents
42 Iajps42102020
42 Iajps42102020
administered one hour before the administration of 2.8. Locomotor Activity (Open Field Test):
CPZ for 5 weeks. Group - I fed with vehicle (Veh) Open field test apparatus is used to measure the
MC suspension which served as normal control. locomotor activity and behavioural responses to a
Group - II, in which MS was induced by new environment. It comes in different sizes, in this
administering CPZ (400 mg/kg, p.o.) served as study we have used a 50 x 50 Cm sized box with dark
disease control. Group - III, in which MS was walls. The box was differentiated into two areas the
induced and treated with the standard drug open arm (central area) and closed arm (area in the
(Fingolimod 0.125 µg/kg, p.o.). Group-IV, in which periphery). The mice were placed in the open arm
MS was induced and treated with the test compound and they are allowed to move freely. The path of the
(Curcumin 200 mg/kg, i.p.). animals was recorded with a video camera placed
above the apparatus. Parameters such as total distance
2.6. Body Weight Monitoring: travelled, average speed was analysed as a measure of
Most of the chemical inducing agents administration locomotor activity with the help of Maze master 1.2.0
can lead to decrease in body weight of animals. To software.
check whether administration of CPZ leads to
decrease in body weight or not the animals were 2.9. Luxol Fast Blue Staining:
weighed daily. Mice were euthanized by cervical dislocation and the
brains were isolated and fixed with 10% neutral
2.7. Biochemical Analysis buffered formalin. The tissues were embedded with
To analyse the protective effects of CCM we paraffin and 8 µm thick sections were cut with the
determined MDA, GSH, levels in homogenates of the help of microtome. The sections were deparaffinized
corpus callosum (CC), which are distinctive for and stained with LFB by incubating them in the
oxidative stress. Mice were transcardially perfused staining solution overnight at 60°C. Following that
with 0.1 M PBS. Following dissection of the brains, sections were differentiated with lithium carbonate
the CCs were isolated. After that, tissue samples were solution, rinsed with 95% and 70% ethanol in a series
homogenized on ice. Tissues homogenates were and then dehydrated with ethanol, mounted with
produced in 0.15 M KCl (5% w/v homogenate). xylene and viewed under the microscope to examine
Microcentrifuge tubes each containing 0.6 mL were the extent of demyelination.
incubated 1 h at 37°C. Following that, 1.2 mL of 28%
w/v trichloroacetic acid (TCA) solution (5%) was 2.9. Statistical analysis:
added, and by adding 1.2 mL of water, the final Results were represented as Mean ± S.E.M., n=6/
volume was made up to 3 mL. Subsequently, group; data was analysed by one-way ANOVA
centrifuged at 3000 x g for 10 min and 2.5 mL of the followed by Tukey’s multiple comparison test using
supernatant was collected. After that, the colour was GraphPad Prism version 8.2.0 (435) software;
developed by addition of 0.5 mL of 1% w/v significance at ***/###p < 0.0001, **/##p < 0.001,
thiobarbituric acid dissolved in 0.05 N NaOH */#p < 0.1. *P vs Veh, # vs Veh + CPZ.
keeping the solution in boiling water bath for 15
minutes until the appearance of pink colour. Finally, 3. RESULTS:
the absorbance was read in a spectrophotometer at 3.1. Effect of CCM on body weight:
532 nm. Malondialdehyde (MDA) contents were Body weight of mice was measured daily throughout
declared as nmol/g wet tissue. The GSH content of the experimental period (Fig 1). During the course
CC was also determined by spectrophotometer vehicle-treated animals showed a slight increase in
according to Sedlak et al.,15 method. Briefly, proteins weight (1.45 ± 5.76). The animals treated with CPZ
from homogenized tissues (10% w/v in PBS, pH 7.4) experienced severe weight loss (-22.33 ± 3.54). The
were removed, after adding an equal volume of 10% animals treated with both CCM (-3.68 ± 5.22) and the
TCA, incubated at 4°C for 2 h. Then, the samples standard drug Fingolimod (-1.12 ± 7.07) were shown
were centrifuged at 2000 x g for 15 min, and the a slight decrease in body weight.
supernatant was added to 2 mL of 0.4 M Tris buffer
(pH 8.9) including 0.02 M EDTA (pH 8.9). After 3.2. Effect of CCM on biochemical parameters:
that, 0.01 M 5,5’-Dithio-bis 2-nitrobenzoic acid was To analyse the effects of CPZ and CCM exposure on
added. Eventually, the mixture was diluted with 0.5 parameters for oxidative stress, we determined MDA
mL distilled water, and absorbance was read in a and GSH levels in homogenates of brain (Fig 2). The
spectrophotometer at 412 nm. Results were shown as results showed that administration of CPZ caused
μg GSH/g wet tissue sample. lipid peroxidation which was evidenced by increase
in MDA levels (30.90 ± 4.36) in CPZ only treated
group, administration of CCM significantly decreased
(18.61 ± 1.20) the lipid peroxidation. Administration 3.4. Luxol fast blue staining:
of CPZ decreased the antioxidant enzyme GSH levels Brain sections of each group were stained with LFB,
(56.91 ± 0.10) in the brain, treatment with CCM in which the lipid rich myelin stains blue and the
significantly attenuates this effect and increased GSH remaining area stands colourless. The myelin density
levels (70.13 ± 0.45) significantly. is evaluated by intensity of colour in the stained
sections, the more the intensity of the stain the lesser
3.3. Open field test: is the demyelination. We concentrated on the corpus
Experimental animals were assessed in the open field callosum region because it is the widely studied
test to evaluate locomotor activity (Fig 3). The total region in demyelination model. LFB staining
distance travelled, average speed was used as a technique was carried out after 5 weeks of treatment
measure of locomotor activity. The CPZ treated protocol. The brain sections of the mice fed with CPZ
animal showed an increase in brain activity which exhibited patches of myelin loss and the colour
was evidenced by increase in locomotor activity intensity was also less when compared to the control
(distance travelled 1371.50 ± 680.75 and average group (Fig 4). The CCM treated group exhibited the
speed 2.93 ± 1.33) treatment with CCM decreased colour intensity similar to that of control group and
(1202.98 ± 554.53, 2.52 ± 0.47) this phenomenon but showed intact myelin.
it was not significant statistically.
10
% Change in body weight
-10
-20
-30
Veh + Veh Veh Fingo CCM
(0.0125 mg/kg p.o.) (200 mg/kg i.p.)
Fig 1: Effect of Curcumin on body weight. The body weight was measured daily during the course, the vehicle
treated group showed slight increase in body weight. The animals administered with the only CPZ experienced
severe weight loss, the animals treated with CCM and Fingolimod weshowed a slight decrease in body weight. It
reveals that CCM attenuates the weight loss caused by CPZ administration
Fig 2: Effect of curcumin on biochemical parameters. The results showed that administration of CPZ caused lipid
peroxidation which increased MDA levels (30.90 ± 4.36) in CPZ treated group. CCM administration significantly
decreased (18.61 ± 1.20) lipid peroxidation. Administration of cuprizone decreased the reduced GSH levels (56.91 ±
0.10) in the brain region, curcumin administration significantly attenuates this effect and increased the levels of
reduced GSH levels (70.13 ± 0.45) significantly.
Fig 3: Effect of curcumin on locomotor activity. The cuprizone treated animals showed an increase in locomotor
which is evidenced by the increase in total distance travelled and speed, treatment with curcumin decreased this
phenomenon but it was not significant statistically.
Fig 4: LFB staining of CC. Demyelination in the CC after 5 weeks of CPZ induction with or without CCM
treatment. Myelin index was evaluated in LFB stained sections of the CC. A) Vehicle, B) CPZ + Vehicle, C) CPZ +
Fingo, D) CPZ + CCM. In the diseased control group the colour was less intense when compared to the vehicle
treated group and showed errosion of myelin. Both the standard drug and the test compound were near to the
intensity of control group and showed intact myelin
4. DISCUSSION:
The present study addresses the anti-oxidant and anti- Multiple sclerosis (MS) is a disease of the central
inflammatory effect of CCM and its protective role in nervous system (CNS) involved with multiple
CPZ induced MS in mice is evidenced by remarkable factors, characterized by inflammation,
reduction of LPO products and increase in reduced demyelination, and axonal loss. It is considered to be
GSH. biphasic disease with inflammatory relapsing-
remitting (RR) and degen-erative secondary
progressive (SP) phases (Silvia BA et al., 2016). neurodegenerative disorders. Mice given with CPZ
The cuprizone model is a well-established instance to for 5 weeks showed increased locomotor activity in
study demyelination and remyelination process in the open field. Administration of CCM reduces the
rodents. Usually, it is administered by mixing with increased CNS activity but it was not statistically
powered or pelleted rodent chow. However, since it is significant. The increase in locomotor activity is
sensitive to the environment and the intake of it related to increased brain activity rather than myelin
varies between different animals, the major issue is damage (Zhang H et al., 2013). The luxol fast blue
the variance in demyelination of the animals. staining of the corpus callosum in the diseased group
Administration of cuprizone at a dose of 400 showed the erosion in white matter, however, the
mg/kg/day by oral gavage was found to be the best fingolimod treated group and CCM treated group
dosage to induce demyelination after 5 weeks of exhibited a similar pattern when compared to the
administration; while remyelination occurs after 9 vehicle treated group.
days of cuprizone withdrawal (Zhen W et al., 2017).
It has an advantage is that the consumption of 5. CONCLUSION:
cuprizone could be well controlled and the mice were In conclusion curcumin at this specified dose level of
exposed to the same dose of cuprizone. Thus, the 200 mg/kg, i.p. in mice has normalized the
variation in demyelination was minimized. This biochemical, behavioural abnormalities in cuprizone
alternative dosing regimen minimizes the inter- induced multiple sclerosis. Further Luxol fast blue
animal variability on demyelination and consequently staining confirmed the neuroprotective activity of
provides a consistent model for pharmacological curcumin in cuprizone induced MS in mice. The
evaluations (Zhen W et al., 2017). actual mechanism of curcumin in cuprizone induced
MS in mice is not clear with these studies. The action
Previous reports have shown that CPZ induces the of curcumin in attenuating the demyelination and
formation of megamitochondria in oligodendrocytes normalizing behavioral changes and other relevant
was determined in CPZ after three weeks. Invitro mediators will be carried out in future to study its
experiments during which primary glial cultures of mechanism.
rats were treated with CPZ provided extra proof that
neuroglia, astrocytes, and oligodendrocytes had 6. Acknowledgement:
shown signs of toxicity upon CPZ treatment (Praet J The author is thankful to Dr. Uday Kiran Juttiga,
et al., 2014). CPZ intoxication clearly increased Consultant, Emergency medicine, Queen Elizabeth
oxidative stress on oligodendrocytes. The formation Hospital, King’s Lynn, UK for his encouragement
of megamitochondria due to CPZ administration during the project.
leads to the shortage of ATP and will increase This work was supported by Shri Vishnu College of
ROS/RNS concentrations of that eventually disrupts Pharmacy, Bhimavaram, West Godavari, A.P, India
the correct functioning of the Endoplasmic reticulum
(ER)17. ER stress additionally reduces mRNA Conflict of interest: The authors report no conflict of
transcription/translation to avoid an accumulation of interest.
un or misfolded proteins. As such, the
downregulation of myelin protein connected mRNAs 7. REFERENCES:
are often determined already at intervals the primary 1. Frohman TC, O’Donoghue DL, Northrop D. A
week of CPZ treatment and continue till CPZ practical primer: Multiple Sclerosis for the
administration is halted (Praet J et al., 2014). Physician Assistant. Dallas, TX: Southwestern
Medical Center. 2011.
CCM suspended in MC administered at a dose of 2. Rosati G. The prevalence of multiple sclerosis in
200mg/kg through intraperitoneal route attenuates the the world: an update. Neurological sciences.
weight loss (-3.68 ± 5.22) occurred due to the toxic 2001 Apr 1;22(2):117-39.
effects of CPZ when compared to diseased group (- 3. Sahoo BM, Banik BK. Medicinal Plants: Source
22.33 ± 3.54). Results have shown that CPZ feeding for Immunosuppressive Agents. Immunol Curr
significantly increased MDA levels (30.90 ± 4.36) Res. 2018;2(106):2.
due to lipid peroxidation, curcumin treatment 4. Johnson JJ, Mukhtar H. Curcumin for
significantly reduced this effect and it also increased chemoprevention of colon cancer. Cancer letters.
the reduced GSH (70.13 ± 0.45) levels when 2007 Oct 8;255(2):170-81.
compared to the diseased group (56.91 ± 0.10). It is 5. Chueh SC, Lai MK, Liu IS, Teng FC, Chen J.
assumed that CCM can restore the normal balance Curcumin enhances the immunosuppressive
between oxidative stress markers and endogenous activity of cyclosporine in rat cardiac allografts
antioxidant mechanisms that is often disrupted in and in mixed lymphocyte reactions.
InTransplantation proceedings 2003 Jun 1 (Vol. neuropathology of the cuprizone mouse model:
35, No. 4, pp. 1603-1605). clinical relevance for multiple sclerosis.
6. Ak T, Gülçin İ. Antioxidant and radical scavenging Neuroscience & Biobehavioral Reviews. 2014
properties of curcumin. Chemico-biological Nov 1;47:485-505.
interactions. 2008 Jul 10;174(1):27-37. 18. Zhang H, Zhang Y, Xu H, Wang L, Zhao J, Wang
7. Julie S, Jurenka M. Anti-inflammatory properties J, Zhang Z, Tan Q, Kong J, Huang Q, Li XM.
of curcumin, a major constituent. Altern Med Locomotor activity and anxiety status, but not
Rev. 2009;14(2):141-53. spatial working memory, are affected in mice
8. Kanakasabai S, Casalini E, Walline CC, Mo C, after brief exposure to cuprizone. Neuroscience
Chearwae W, Bright JJ. Differential regulation of bulletin. 2013 Oct 1;29(5):633-41.
CD4+ T helper cell responses by curcumin in 19. Slowik A, Schmidt T, Beyer C, Amor S, Clarner
experimental autoimmune encephalomyelitis. T, Kipp M. The sphingosine 1‐phosphate
The Journal of nutritional biochemistry. 2012 receptor agonist FTY 720 is neuroprotective after
Nov 1;23(11):1498-507. cuprizone‐induced CNS demyelination. British
9. Zhen W, Liu A, Lu J, Zhang W, Tattersall D, journal of pharmacology. 2015 Jan;172(1):80-92.
Wang J. An alternative cuprizone-induced 20. Goldenberg MM. Multiple sclerosis review.
demyelination and remyelination mouse model. Pharmacy and Therapeutics. 2012
ASN neuro. 2017 Aug;9(4):1759091417725174. Mar;37(3):175.
10. McCarthy DP, Richards MH, Miller SD. Mouse 21. Reddy PH, Manczak M, Yin X, Grady MC,
models of multiple sclerosis: experimental Mitchell A, Kandimalla R, Kuruva CS.
autoimmune encephalomyelitis and Theiler’s Protective effects of a natural product, curcumin,
virus-induced demyelinating disease. against amyloid β induced mitochondrial and
InAutoimmunity 2012 (pp. 381-401). Humana synaptic toxicities in Alzheimer's disease.
Press, Totowa, NJ. Journal of Investigative Medicine. 2016 Dec
11. Taylor LC, Gilmore W, Matsushima GK. SJL 1;64(8):1220-34.
mice exposed to cuprizone intoxication reveal 22. Seibenhener ML, Wooten MC. Use of the open
strain and gender pattern differences in field maze to measure locomotor and anxiety-
demyelination. Brain Pathology. 2009 like behavior in mice. JoVE (Journal of
Jul;19(3):467-79. Visualized Experiments). 2015 Feb
12. Taylor LC, Gilmore W, Ting JP, Matsushima 6(96):e52434.
GK. Cuprizone induces similar demyelination in 23. Rao MN. Curcuminoids as potent inhibitors of
male and female C57BL/6 mice and results in lipid peroxidation. Journal of Pharmacy and
disruption of the estrous cycle. Journal of Pharmacology. 1994 Dec;46(12):1013-6.
neuroscience research. 2010 Feb 1;88(2):391- 24. Elmegeed GA, Ahmed HH, Hashash MA, Abd-
402. Elhalim MM, El-Kady DS. Synthesis of novel
13. Sajjadian M, Kashani IR, Pasbakhsh P, Hassani steroidal curcumin derivatives as anti-
M, Omidi A, Takzare N, Clarner T, Beyer C, Alzheimer’s disease candidates: Evidences-based
Zendedel A. Protective effects of cannabidiol on on in vivo study. Steroids. 2015 Sep 1;101:78-
cuprizone-induced demyelination in C57BL/6 89.
mice. Journal of Contemporary Medical 25. Girish C, Koner BC, Jayanthi S, Ramachandra
Sciences. 2017 Sep 26;3(11):278-83. Rao K, Rajesh B, Pradhan SC. Hepatoprotective
14. Prasad S, Tyagi AK, Aggarwal BB. Recent activity of picroliv, curcumin and ellagic acid
developments in delivery, bioavailability, compared to silymarin on paracetamol induced
absorption and metabolism of curcumin: the liver toxicity in mice. Fundamental & clinical
golden pigment from golden spice. Cancer pharmacology. 2009 Dec;23(6):735-45.
research and treatment: official journal of Korean 26. Chandra D, Gupta SS. Anti-inflammatory and
Cancer Association. 2014 Jan;46(1):2. anti-arthritic activity of volatile oil of Curcuma
15. Sedlak J, Lindsay RH. Estimation of total, longa (Haldi). The Indian journal of medical
protein-bound, and nonprotein sulfhydryl groups research. 1972 Jan;60(1):138.
in tissue with Ellman's reagent. Analytical 27. Aggarwal ML, Chacko KM, Kuruvilla BT.
biochemistry. 1968 Jan 1;25:192-205. Systematic and comprehensive investigation of
16. Silva BA, Ferrari CC. Animal experimental the toxicity of curcuminoid-essential oil
models for understanding and treating Multiple complex: A bioavailable turmeric formulation.
Sclerosis. Molecular Medicine Reports. 2016 Jan
17. Praet J, Guglielmetti C, Berneman Z, Van der 1;13(1):592-604.
Linden A, Ponsaerts P. Cellular and molecular
28. Browning V, Joseph M, Sedrak M. CME 33. Poorani ES, Brundha MT. Preservative and
ARTICLE Multiple sclerosis: A comprehensive Fixative Methods of Brain Biopsy-Review.
review for the physician assistant. Journal of the Journal of Pharmaceutical Sciences and
American Academy of PAs. 2012 Aug Research. 2015 Jun 1;7(6):336.
1;25(8):24-9. 34. Prasad S, Tyagi AK, Aggarwal BB. Recent
29. Franco-Pons N, Torrente M, Colomina MT, developments in delivery, bioavailability,
Vilella E. Behavioral deficits in the cuprizone- absorption and metabolism of curcumin: the
induced murine model of golden pigment from golden spice. Cancer
demyelination/remyelination. Toxicology letters. research and treatment: official journal of Korean
2007 Mar 30;169(3):205-13. Cancer Association. 2014 Jan;46(1):2.
30. Gudi V, Gingele S, Skripuletz T, Stangel M. Glial 35. Qureshi M, Al-Suhaimi EA, Wahid F, Shehzad
response during cuprizone-induced de-and O, Shehzad A. Therapeutic potential of curcumin
remyelination in the CNS: lessons learned. for multiple sclerosis. Neurological Sciences.
Frontiers in cellular neuroscience. 2014 Mar 2018 Feb 1;39(2):207-14.
13;8:73. 36. Sriram S, Steiner I. Experimental allergic
31. Liu W, Zhai Y, Heng X, Che FY, Chen W, Sun encephalomyelitis: a misleading model of
D, Zhai G. Oral bioavailability of curcumin: multiple sclerosis. Annals of Neurology: Official
problems and advancements. Journal of drug Journal of the American Neurological
targeting. 2016 Sep 13;24(8):694-702. Association and the Child Neurology Society.
32. MacKenzie‐Graham A, Lee EF, Dinov ID, Bota 2005 Dec;58(6):939-45.
M, Shattuck DW, Ruffins S, Yuan H, 37. Frohman TC, O’Donoghue DL, Northrop D. A
Konstantinidis F, Pitiot A, Ding Y, Hu G. A practical primer: Multiple Sclerosis for the
multimodal, multidimensional atlas of the Physician Assistant. Dallas, TX: Southwestern
C57BL/6J mouse brain. Journal of anatomy. Medical Center. 2011.
2004 Feb;204(2):93-102.