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Evaluation of Carvedilol Effects on Scopolamine Induced -Alzheimer Model1. Research Problem:Alzheimer disease (AD) is one of the most devastating neurodegenerative disease that present with cognitive and memory skills impairment. It is now recognized as one of the most world widely neurodegenerative disorders and it is recorded for more than eighty percent of dementia cases in elderly patients(Anand, Gill, & Mahdi, 2014). Approximately two hundred – thousand people younger than sixty-five years with AD considered as the younger onset phase of AD population; five million are sixty-five or older.
It is estimated that by 2050, every thirty three second, one case of AD will develop, or approximately a million new cases yearly, and the total calculable prevalence is predicted to be 13.8 million (Association, 2016).The AD pathophysiology includes different particular and molecular pathways of neuronal destruction, oxidative stress and inflammation, in which the damage of cholinergic neurons is the main one. This has been long attributed to pathological deposition of amyloid peptides and formation of neurofibrillary tangles constituted by hyper-phosphorylated aggregates of the microtubule-associated protein, tau(Mostafa, Ismail, & Ghareeb, 2016).
Nowadays, the approved Alzheimer therapeutic medication by US Food and Drug Administration (FDA), are mainly 5 medications that are used for symptomatic treatment for cognitive impairments of AD, acetyl choline esterase inhibitors”rivastigmine, galantamine , tacrine, and donepezil and NMDA receptor antagonist(memantine) (Kumar, Prakash, & Dogra, 2011). Each drug has a different mechanism of action to inhibit the breakdown of acetylcholine (important chemical neurotransmitter in the brain for memory function). Tacrine is not prescribed because it has serious hepatotoxicity features.
Generally, rivastigmine, galantamine, and donepezil are most effective medications for the treatment early stages of Alzheimer disease. Memantine is the only drug that show beneficial effects on the late stages of (AD). All of these medications are working by slow the progression of cognitive impairment symptoms, but at least half of AD patients who on these treatments do not respond to them(Mostafa et al., 2016). These therapeutic strategies only postpone the progression of the manifestation associated with AD. Much effort is needed to be directed towards the discovery of new disease-modifying treatments which can inhibit the progression of the disease by targeting other pathological mechanism as oxidative stress.Carvedilol is a nonselective І -adrenergic receptor blocker, it has been used for the treatment of hypertension and congestive heart failure (Packer et al., 1996). It was suggested that carvedilol show a specific unique three-dimensional pharmacophore conformation, associated with the ability to bind amyloid bets (AІ) and prevent AІ from forming oligomeric neuro- fibrils (Wang et al., 2014). Another recent study suggested that use of carvedilol is strongly related to cognitive beneficial effects in AD people due to anti- oxidative and anti-inflammatory features (Rosenberg et al., 2008).2. ObjectiveTherefore, this study will be conducted to examine carvedilol effects on learning and memory behavior in a rat model of cognitive impairment induced by scopolamine, a muscarinic antagonist. The study, also, aimed to investigate the mechanisms underlying carvedilol effects, whether they are related to the cholinergic pathway, modulation of associated inflammatory and oxidative stress mechanism. 3. Importance of researchIn relation to the cholinergic deficit, pharmacological model of scopolamine induced amnesia considered as a good represented model for cognitive impairment. Scopolamine causes alterations of the cholinergic pathways in the neurons and memory mechanism in the brain. In addition, there is a research reported scopolamine as an agent affect many genetic expression that related to some important pathways, such as apoptosis, cytoskeleton reconstruction, protein trafficking and cell differentiation (Hsieh, Hsieh, Lin, Wu, & Huang, 2003). These dysregulations lead to serious impairment in memory skills simulating Alzheimer disease cholinergic deficits (Lee, Sur, Shim, Lee, & Hahm, 2012).Furthermore, the AD pathology includes various particular and overlapping mechanisms of neuronal damage, apoptosis, aggravation and oxidative stress. Focusing on new pathways to develop new medication altering other mechanisms is a major goal in the management of Alzheimer disease. Numerous investigations demonstrate the antioxidant and anti-inflammatory effects of carvedilol which may give a reasonable connection of its neuroprotective impacts in AD.4. Literature reviewAlzheimer disease (AD) is one of the most devastating progressive degenerative disease that affects nervous system. It is presenting mainly by cognitive and memory skills impairment. It has a major two phases which are, preclinical (pre-symptomatic phase) and clinical (symptomatic) phase. Another way for classification is to subcategorized into mild cognitive impairment (MCI) due to AD (prodromal AD) and AD dementia with mild, moderate, and finally severe late stages (Epelbaum et al., 2017). Main neuropathological sings of Alzheimer disorder are amyloid plaques(AІ) present extra-cellularly and neuro-fiber tangles present intracellular in the brain of Alzheimer patients (Serrano-Pozo, Frosch, Masliah, & Hyman, 2011). Besides these special characters of Alzheimer disorder, a large research indicates and report the presence of a number of other pathophysiological mechanisms which are common for many neurodegenerative disorders and for aging process of normal brain, reviewed in (Molteni & Rossetti, 2017), These include permanent activation of inflammatory and immunological mediators , mitochondrial damage and iron disposition (Ayton, Lei, & Bush, 2013). The main cause of Alzheimer disorder is incompletely understood. Most of the Alzheimer patients are late onset, with most likely extremely complex cause, influenced genetic factors (Rosenberg et al., 2008), environmental risk , aging which considered the most important risk factor (Cuyvers & Sleegers, 2016; Killin, Starr, Shiue, & Russ, 2016).The identification of amyloid plaques in some areas in the brain of Alzheimer patients and the detection of pathogenic changes in APP and PSEN1 genes have led to the development of the amyloid plaques hypothesis, which supports that AІ is the fundamental change in Alzheimer pathogenesis . Nevertheless, further investigations have exhibited that in spite of the fact that AІ formation is essential for the development of Alzheimer disorder, it isn’t adequate, since the AІ pathology can exist with the absence of clinical dementia . Moreover, many clinical studies targeting amyloid plaques failed to provide clinical advantages in Alzheimer treatment (Hyman & Sorger, 2014). These results have led to the reformulation the hypothesis of amyloid cascade in Alzheimer disorder and development other new theories, including the mitochondrial damage, inflammation and oxidative stress hypotheses, which propose the role free radicals theory with aging in Alzheimer disease(HARMAN, 1956) .Expanding evidence has supports that Carvedilol has neuroprotective impacts due to its antioxidant property. Carvedilol provides a protection to PC12 cells against 6- OHDA-induced neural damage possibly by raising the viability of the cells, decreasing reactive free radicals and oxygen species , and stimulating the Akt and Nrf2/ARE signaling mechanisms (Wang et al., 2014). Another investigation exhibited the neuroprotective capability of Carvedilol in aluminum chloride-induced Alzheimer model and (also caused oxidative damage) by long term administration of Carvedilol daily to rats for a period of one month and a half, significantly enhanced the memory skills and rats performance tasks in the Morris water maze test, decrease the oxidative stress (by reduced lipid peroxidation, nitrite concentration and restored reduced glutathione, superoxide dismutase, catalase, and glutathione S-transferase activity), decreased activity of acetylcholinesterase enzyme, and concentration of aluminum in treated rats group compared to control rats (Kumar et al., 2011).Additionally, there was a research supported the use of Carvedilol in Alzheimer patients is strongly related to a cognitive benefit (Rosenberg et al., 2008).Interestingly, a recent research examined whether Carvedilol has a protective impact on the neurons against endogenous amyloid plaques in mouse Neuro2a (N2a) cells transfected with Swedish amyloid precursor protein (Swe-APP) mutant and Presenilin exon9 deletion mutant (N2a/Swe.D9). In N2a/Swe.D9 cells, the levels of ATP and mitochondrial membrane potential were decreased, which were restored by treatment with Carvedilol. N2a/Swe.D9 cells displayed increased vulnerability to H2O2-induced cell death and apoptosis, whichcould be significantly reduced by Carvedilol. Mechanistically, it was documented that Carvedilol reduce the release of cytochrome C and the level of cleaved caspase-3, which results in the prevention of apoptosis. 5. Research Methodology and ProceduresAnimalsExperiments will be carried on 40 adult Wistar rats weighing 200-250 g. The rats will behoused under standard research lab facility conditions with free access to standard food and water, and will be left to adjust for many weeks. Every one of the trials will be held in specific place in the range of 9:00 a.m. and 3:00 p.m. Standards of research center for animal care will pursued, and the convention was endorsed by the Ethics Committee of Faculty of Medicine, King Abdelaziz University.Drugs and chemicalsScopolamine hydrobromide, carvedilol will be obtained from Novartis Pharma (Jeddah,SA), All other chemicals were commercial products. Experimental design for inducing memory and cognitive impairment, treated rats will receive an intraperitoneal injection of scopolamine prepared solution (dissolved in normal saline NACL 0.9% with a final concentration of 200 јg/ml ) in a dose of 1 mg/kg/day once daily for 15 days, thirty min before any behavior test (Moosavi, Khales, Abbasi, Zarifkar, & Rastegar, 2012). Control rats will receive an equal volume of NS 0.9% solution instead. Accordingly, the rats will be assigned into 4 groups of 10 rats each: NS 0.9%-treated control group, scopolamine treated group, carvedilol- treated (10 јM/kg/day) scopolamine group, carvedilol- treated (20 јM/kg/day) scopolamine group. Carvedilol or normal saline will be given 60 min before scopolamine intraperitoneal injection throughout the experiment.Evaluation of memory activitiesAcquisition and maintenance of conditioning-based memory skills will be tested byMorris water maze (MWM) and passive avoidance tasks. To avoid rat exhaustion and confounding memory behavior, different animals in each treated group will subjected to either Morris water maze or passive avoidance tasks.Morris water maze taskThe rats will be prepared to swim to a stage in a round pool (180 cm distance across — 60cm tallness) loaded up with water (22 ± 2 °C) to a profundity of 40 cm. The pool was basically isolated into four equivalent quadrants: north, south, east and west. A white stage 15 cm in distance across was submerged roughly 2 cm underneath the water surface, in the focal point of one of the four quadrants all through the errand. To conceal the stage, the water was made hazy by including milk powder. Obvious signals were settled outside the pool helping the animals finding the concealed stage. A settled computerized camera was utilized to record rats movement all through the maze. The MWM included three distinct tests:Hidden platform testThis test will be performed for 4 consecutive days, beginning on the tenth day ofscopolamine infusion, where six rats per treated group got instructional sessions comprising of two session per day with 10-min of each. Each session comprised of four trials with 4 different rats on different beginning positions. The normal time taken by a rat to achieve the stage, in the first day, was recorded as initial acquisition latency up to a greatest of 90 s, while those of thesecond, third and fourth days were recorded as escape retention latencies, reflecting retention of learned tasks.Probe testOn day 15, the probe test will be performed after removal of the platform. It consisted ofa one minute free swimming time, and the period spent in the quadrant of the removed platform will be documented. It is an estimation for the strength and accuracy of spatial memory skills.Visible platform testThis test is mainly to measure the vision acuity of the rats. Immediately after the probetest, the visual signs will be removed and the platform was made visible by a lightreflective cover that raises up over the water surface. Two trials will be taken into account that each rat can find the stage; there was no time limit in this trial, a half minute lay was permitted on the stage and after that any latency will be evaluated in the second trial. This test is important to reject any iatrogenic changes in the rat’s visual accuracy.Locomotor activityPreceding the MWM test, the rats voluntary motor skills will be evaluated in an openarea to exclude the scopolamine impacts on rat’s motor activities. The open area is a box made from wood material with one wall of glass, and its floor is divided into equivalent measured squares. Each rat will be observed for five minutes; then based on that, a grade for total locomotor activities determined as the total of line crosses and rears .Passive avoidance taskOn days ten and eleven after the beginning of scopolamine injection, the six rats pergroup will be trained in a passive avoidance device. It comprises of 2 separate illuminated dark chambers (22 — 21 — 22 cm each), connected by a door (8 — 15 cm). The dark floor of the chamber special type of steel that deliver electric shocks (0.5 mA). The rats on acquisition trial, should be trained to avoid that place, because it is related with aversive event .That will be achieved by two phases: the first one is a pre-acquisition trial, each rat will be placed in the illuminated chamber, and back to the door; then after half a minute of habituation, the door will be opened and the rat will be permitted to enter the dark one freely. The same thing will be repeated for fifteen min later in the acquisition trial, where the initial step-through latency to enter the dark chamber was documented. Once the animal go to the dark chamber completely, the door will closed electric shock will be delivered for 3 seconds ; then, it will returned to its home. Any rat will fail to enter the dark chamber within five minutes will be excluded from the experiment and will substituted by another one from the group. Following day, the maintenance or latency time to enter the dark chamber will documented to a greatest of five minutes without electric shock. The decline in latency time is an indicator of a memory deficit in the passive avoidance task .One of the limitations of this research is that only two doses of carvedilol will be evaluated. It would be very interesting to study the neuro-protective effects of a different doses of carvedilol on other aspects of Alzheimer disorder pathophysiology, particularly AІ and secretases pathways, and neuronal cell death . 6. Research Limitation 7. Study’s overall structure Activity No. Tasks Months 1 Approval and fund 2 2 Field work 4 3 Data Analysis 2 4 Report writing 2 5 Submitting report 2 8. Expected Results:Carvedilol , will offers a neuro- protective effects against scopolamine-induced cognitive impairment, and show an improvement on cognitive and memory skills in Morris water maze task And passive avoidance tests. These effects may change according to the dose range.9. TerminologyCognitive impairment .carvidelol . Scopolamine .Phosphorylated tau . cholinesterase activity ,AD Alzheimer’s disease , AChE Acetyl cholinesterase, ,MWM Morris water maze , AІ Amyloid beta , passive avoidance test . 10. ReferencesAnand, R., Gill, K. D., & Mahdi, A. A. (2014). Therapeutics of Alzheimer’s disease: Past, present and future. Neuropharmacology, 76 Pt A, 27-50.Association, A. s. (2016). 2016 Alzheimer’s disease facts and figures. Alzheimers Dement, 12(4), 459-509.Ayton, S., Lei, P., & Bush, A. I. (2013). Metallostasis in Alzheimer’s disease. Free Radic Biol Med, 62, 76-89.Cuyvers, E., & Sleegers, K. (2016). 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