Ms Blessina Sugandhi Tutor Department of Anatomy, YMC .

1. Morphological, histological changes and estimation of Biomarker Aβ-42 in the Brain of Alzheimer's disease rats; modification by Valeric acid Research Student • Guide • Dr.Bindhu. S • Associate Professor • Department of Anatomy,YMC. • Ms BlessinaSugandhi • Tutor • Department of Anatomy, YMC.

2. CONTENTS • Introduction • Literature Review • Lacunae in the literature • Relevance of my study • Aim and Objectives • Materials & Methodology • Statistical analysis • Budget plan • Timeline • References Pre PhD Synopsis

3. INTRODUCTION • Memory is the ability of an individual to record sensory stimuli, events, information, etc (Rahman H, 2011) • Central cholinergic system, plays an important role in learning and memory (Blokland A, 1995) • Alzheimer’s disease (AD) is associated with a progressive loss of cholinergic neurons and a decline in the levels of acetylcholine in the brain (Blokland A, 1995) Pre PhD Synopsis

4. INTRODUCTION • Valeric acid is a straight-chain alkyl carboxylic acid with the chemical formula C5H10O2. It is a colourless liquid found naturally in the perennial flowering plant Valerianaofficinalis, from which it gets its name(Merck Index, 12th Edition, 10042) • Although it lacks the alcohol and amine functional groups that contribute to the biological activities of GHB and GABA; (Merck Index, 12th Edition, 10042) • Valeric acid is structurally similar to the neurotransmitter, GABA (Grossman L, 2011) Pre PhD Synopsis

5. Review Literature Pre PhD Synopsis

6. Review Literature Pre PhD Synopsis

7. LACUNAE IN THE LITERATURE • The neurological processes that leads to Alzheimer’s disease is multi-factorial • Memory enhancing drugs are costly and have minimal efficacy • An alternative agent which is more efficacious as well as economical is yet to be discovered Pre PhD Synopsis

8. RELEVANCE OF THE STUDY • Increasing incidence of Alzheimer’s disease is a social concern and financial burden for the society • This pre-clinical study is focused to develop a better drug for AD over the existing drugs • This study will prove beneficiary to the people suffering from AD worldwide Pre PhD Synopsis

9. AIM • To study the effect of Valeric acid on morpho-histometric changes, immuno-histochemical changes, neurotransmitter and Aβ- 42 levels in the brains of Alzheimer’s disease induced rats Pre PhD Synopsis

10. OBJECTIVES • To study the memory enhancing activity of Valeric acid in Alzheimer’s disease rats through pharmacological screening • To study the morphological and histological changes in the brains of Valeric acid treated Alzheimer’s diseased rats • To document the degree of damage of brain tissues by immuno-histochemical analysis • To study the effect of valeric acid on neurotransmitter levels in the brains of Alzheimer’s diseased rats in comparison with the untreated ones • To study the effect of valeric acid on Aβ- 42 levels in the brains of Alzheimer’s diseased rats in comparison with the untreated ones Pre PhD Synopsis

11. Methodology • The study will be initiated after getting clearance from Institutional Animal Ethics Committee (IAEC) Pre PhD Synopsis

12. Phases of the experimental work Phase 1:Toxicity Studies • Acute and Chronic toxicity studies of Valeric acid Phase 2:Evaluation of therapeutic role of Valeric acid in AD • Phase 2a: Inducing of AD using aluminum chloride • Phase 2b: Evaluation of protective role of Valeric acid, Piracetam and Rivastigmine Pre PhD Synopsis

13. Phases of the experimental work • Phase 1:Toxicity Studies • Phase 1a: Acute Toxicity of Valeric acid n=5 for each test compound Dose: 2000mg/kg body wt. orally Animals will be observed for signs of toxicity from 0th hour of dosing to 14th day to determine the dose of test compounds. Pre PhD Synopsis

14. Phases of the experimental work • Phase 1:Toxicity Studies • Phase 1b: Chronic Toxicity of Valeric acid n=6 for each test compound Dose: therapeutic dose as determined by LD50 study will be administered orally for 60 days Parameters observed: 1. Behavioral changes 2. Body weight 3. Hepatic Function 4. Renal function 5. Histological changes in vital organs Pre PhD Synopsis

15. Phases of the experimental work • Phase 2:Evaluation of therapeutic role of Valeric acid • Appropriate animals (Wistar albino rats) will be selected for the study • The animals will be divided into 7 different groups. (Each group consisting of 6 animals, 03 males & 03 females.) • a) Normal control (Distilled water) , • b) Aluminum chloride(70mg/kg) (Ali A., 2016) • c) AD + Piracetam(200mg/kg) (Ali A., 2016) • d) AD + Rivastigmine(0.5 mg/kg) (Ali A., 2016) • e) AD + Valericacid (dose based on LD50 study) • f) AD + Piracetam + Valeric acid • g)AD + Rivastigmine + Valeric acid Pre PhD Synopsis

16. All of them will be administered with AlCl3 (70mg/kg i.p) (Ali A., 2016) to induce Alzheimer’s disease • Once the rats develop Alzheimer’s disease(which will be confirmed by EPM test), drugs/vehicle will be administered to the respective animals • After which, the animals will be screened to assess the role of drugs on memory • Once the memory enhancing activity is established, the animals will be euthanized using ketamine. Brains will be dissected out to carry out biochemical and histological studies Pre PhD Synopsis

17. Materials • Aluminum chloride salt • Valeric acid solution • Piracetam tablets • Rivastigmine tablets • Animals required • Species: Wistar albino rats • Age/ weight/ Gender: 4-6 months; 200g of both sex • Number of animals required : 53 Animals • Number of days each animals will be housed: 75 days Pre PhD Synopsis

18. Table 1. Animal study plan. Pre PhD Synopsis

19. Assessment of Memory • Elevated plus maze (EPM) Figure 2. Assessment of memory Pre PhD Synopsis

20. Morphological changes • Decrease in the gray matter volumes in the medial temporal structure, hippocampus • Altered medial temporal structures(Ohnishi T, 2001) Pre PhD Synopsis

21. Histological changes • After the administration of drugs , the brains of the rats will be withdrawn and fixed with para-formaldehyde and then after histological processing, the slices will be stained with acid haematoxylin for cells of hippocampus.(Ohnishi T, 2001) • These sections will be compared with sections of control group • Examined for Hirano bodies Neuro-fibrillary tangles Senile plaques Pre PhD Synopsis

22. Immunohistochemistry • Brain  tissues will be fixed in 10% neutral-buffered formalin solution, dehydrated in graded alcohol, and then embedded in paraffin. (Hashem HE., 2010) • The brain tissue sections will be stained with heamatoxylin and eosin. • The degree of damage  will be assessed semi quantitatively at 400X magnification using 20 randomly selected fields under a light microscope. Pre PhD Synopsis

23. Estimation of Aβ-42 Biomarker • AD is characterized by extracellular Aβ plaque depositions. • In brain tissue homogenate, determination of Aβ content will be assessed using ELISA Kits, according to the manufacturer’s instructions (Ali A., 2016) Pre PhD Synopsis

24. ESTIMATION OF NEUROTRANSMITTERS (RahmanH et al., 2013) 1. Estimation of Dopamine The dopamine content will be estimated by spectrofluorimeter 2. Estimation of Serotonin The serotonin content will be estimated by spectrofluorimeter 3. Estimation of Glutamate and GABA levels The level of Glutamate and GABA will be estimated by multiple development paper chromatography method Pre PhD Synopsis

25. ACETYLCHOLINESTERASE ENZYME DETERMINATION Acetylcholinesterase (AChE) enzyme activity will be estimated using spectrophotometer (RahmanH et al., 2013) Pre PhD Synopsis

26. STATISTICAL ANALYSIS All the groups will be subjected to analysis of variance by using one-way ANOVA followed by Tukeykrammer test. Pre PhD Synopsis

27. Timeline Pre PhD Synopsis

28. Budget plan Pre PhD Synopsis

29. References • Rahman H, Shaik HA, Madhavi P, Eswaraiah MC. A review: pharmacognostics and pharmacological profiles of Nardastachys jatamansi DC. Elixir pharmacy. 2011 Sep 23;39:5017-20. • Blokland A. Acetylcholine: a neurotransmitter for learning and memory?. Brain Research Reviews. 1995 Nov 30;21(3):285-300. • Grossman L, Stewart A, Gaikwad S, Utterback E, Wu N, DiLeo J, Frank K, Hart P, Howard H, Kalueff AV. Effects of piracetam on behavior and memory in adult zebrafish. Brain research bulletin. 2011 Apr 25;85(1):58-63. • Ali AA, Ahmed HI, Abu-Elfotuh K. Modeling Stages Mimic Alzheimer’s Disease Induced by Different Doses of Aluminum in Rats: Focus on Progression of the Disease in Response to Time. of. 2016;11:2. • Sadigh-Eteghad S, Sabermarouf B, Majdi A, Talebi M, Farhoudi M, Mahmoudi J. Amyloid-beta: a crucial factor in Alzheimer's disease. Medical principles and practice. 2015;24(1):1-0. • Prince M, Ali GC, Guerchet M, Prina AM, Albanese E, Wu YT. Recent global trends in the prevalence and incidence of dementia, and survival with dementia. Alzheimer's Research & Therapy. 2016 Jul 30;8(1):23. • Gopalakrishnan K, 2016, Alzheimer’s Related & Disorders Society of India. Pre PhD Synopsis

30. References • Whitehouse PJ. The end of Alzheimer's disease—From biochemical pharmacology to ecopsychosociology: A personal perspective. Biochemical pharmacology. 2014 Apr 15;88(4):677-81. • Rao RV, Descamps O, John V, Bredesen DE. Ayurvedic medicinal plants for Alzheimer's disease: a review. Alzheimer's research & therapy. 2012 Jun 29;4(3):1.1. Birks JS, Grimley Evans J. Rivastigmine for Alzheimer's disease. The Cochrane Library. 2015. • Achliya GS, Barabde U, Wadodkar S, Dorle A. Effect of BramhiGhrita, an polyherbal formulation on learning and memory paradigms in experimental animals. Indian Journal of Pharmacology. 2004 May 1;36(3):159. • Joshi H, Parle M. Nardostachysjatamansi improves learning and memory in mice. Journal of medicinal food. 2006 Mar 1;9(1):113-8. • Vishwakarma S, Goyal R, Gupta V, Dhar KL. GABAergic effect of valeric acid from Valerianawallichii in amelioration of ICV STZ induced dementia in rats. RevistaBrasileira de Farmacognosia. 2016 Aug 31;26(4):484-9. • Hashem HE, Elmasry SM, Eladl MA. Dentate gyrus in aged male albino rats (histological and Tau-immunohistochemical study). Egypt J Histol. 2010;33:659-70. Pre PhD Synopsis