Curcumin, FUS, and AD: When can the world finally forget about Alzheimers?

Aadya Sharma

Independent Research

05/10/2017

Advisor: Me Kim

Ms. Julia Bakhru

When Can the World Finally Forget About Alzheimers?

Aadya Sharma

May 2017
 1

Advisor: Me Kim

The general area of research for this project is why curcumin has not been implemented

as a cognition-improving drug in humans, how the BBB impedes drug delivery to the brain, and

how Focused Ultrasound Therapy (FUS) can disrupt this barrier to allow otherwise barricaded

molecules to pass into the brain. A number of authentic studies about the applications of FUS, the

resulting disruption of the BBB, and the ability of curcumin to improve cognition will be

analyzed, cross-referenced, and evaluated to determine whether FUS can effectively improve the

bioavailability of curcumin in patients with Alzheimers, thereby creating a possible early-stage

preventative for the progression of the disease. A short film will then be produced by the

researcher to showcase and propagate the findings to scientists and those who are affected

directly or indirectly by Alzheimers to inform them of possible preventative options.

Introduction:

After the age of 85, half of the population will suffer from Alzheimers Disease (AD).

This trend, however, can be changed. Derivatives of the anti-inflammatory drug curcmin have

May 2017
 2

been shown to reverse some of the symptoms of Alzheimers in mice models of the disease

(Prior Marguerite, et al. 2013)[14]. In fact, Alzheimers has lower prevalence and incidence in

regions where turmeric, the source of curcumin, is used as a staple herb (Prasad Sahdeo and

Bharat B. Aggarwal 1970; Prince, Martin, et al. 2015, 10)[17],[15]. The drug, however, has not been

implemented successfully in humans primarily because of it inability to traverse the Blood Brain

Barrier (BBB) that encases and protects blood vessels in the brain. To combat this issue, Focused

Ultrasound Therapy (FUS) can be used to facilitate the delivery of curcumin to the brain. Due to

its ability to temporarily disrupt the Blood Brain Barrier, the application of FUS is crucial for

enhancing the bioavailability of curcumin as a cognition-improving drug for patients with

Alzheimers. The purpose of this essay is to explain the role of inflammation in Alzheimers,

analyze the effectiveness of curcumin as a cognition-improving drug, elaborate on the role

played by the BBB, and justify the use of FUS. Additionally, it will provide suggestions for other

techniques that can be coupled with FUS to further improve the bioavailability of curcumin.

Review of Literature:

Alzheimers is a chronic neurodegenerative disease characterized by cognitive

impairment, dementia, and paralysis. It was first scientifically discovered in 1906 by Doctor

Alois Alzheimer when he observed unusual markings in the brain of his senile patient, Auguste

Deter (Alzheimers Association)[3]. Over the course of a century, scientists have researched this

mysterious disease and have proposed several theories as to what its cause might be. The most

recent one attempts to explicate the relationship between beta-amyloid plaques and the

pathogenesis of Alzheimers. It has been suspected that clumps of proteins, beta-amyloid

plaques, called protein aggregates cluster throughout the brain and lead to neuronal death. This

initiates a cascade effect that leads to cognitive impairment, significant memory loss, dementia

May 2017
 3

and immobility (Stevens 2016)[19]. Additionally, neurofibrillary tangles, such as tau protein

tangles, within neurons impair nutrient flow, thereby contributing to neuronal death (Stevens

2016)[19]. The beta amyloid hypothesis has led several pharmaceutical companies, including

Biogen and Eli Lilly, to formulate and experiment drugs targeting beta amyloid in the brain, but

the unfortunate lack of success of these drug trials, a 99.6% failure rate, has led scientists to

reexamine the beta amyloid hypothesis and target other possible contributors to the disease, such

as inflammation (Alzheimers Association)[3].

Inflammation is the natural response of living tissues to injuries, in which parts of the

body become reddened, swollen, hot and painful. In essence, it is the bodys attempt at self-

protection by removing harmful stimuli. Inflammation has been suspected to be both a symptom

and a cause of Alzheimers by many scientists. In fact, through multiple immunohistochemical,

biochemical, and molecular studies, it has been shown that there are many inflammatory

mediators in the Alzheimers brain (Akiyama, Haruhiko, et al)[1], such as microglia. Microglia

are cells in the Central Nervous System (CNS) that function as macrophages, consuming foreign

substances in the CNS. Their activation appears to be the most prominent inflammatory

component in chronic inflammatory diseases (Streit, Wolfgang J., et al. 2004)[20]. Alzheimers is

believed to be one such inflammatory disease in which beta amyloid plaques in the brain are the

center of microglia activation and clustering (Streit, Wolfgang J., et al. 2004)[20]. The chronic

inflammation associated with activated microglia is believed to correlate with cognitive

impairment. In fact, a Harvard University research groups study stated that the elimination of

amyloid plaques might not be the optimal method to treat Alzheimers, and instead, treating

inflammation may be crucial in preventing or reducing the progression of the disease (Weintraub

2016)[23]. Another study led by Johns Hopkins University Professor Michela Gallagher found that

May 2017
 4

reducing neural hyperactivity in patients with amnestic Mild Cognitive Impairment (aMCI) using

the epilepsy drug levetiracetam improved the patient's cognitive function (Gallagher 2011)[6].

However, these anti-inflammatory/hyperactivity medications often have low success rates,

discouraging patients from receiving any treatment. Curcumin appears to be the potent and

efficient, not to mention safer, natural anti-inflammatory alternative to these drugs.

Studies, reviews and historical usage of turmeric attest to its remarkable abilities as an

anti-inflammatory drug, the reason being that the herb contains chemicals called curcuminoids,

the active ingredients in turmeric. The most important of these curcuminoids is curcumin itself.

Curcumin is a drug with high levels of antioxidant and anti-inflammatory properties, including

blocking inflammatory molecules and boosting levels of BDNF (Brain Derived Neurotrophic

Factor Proteins) in the brain. These proteins are the caretakers of neurons, they protect them from

harmful chemicals, promote their growth, connection, and the formation of memories (Prior,

Marguerite, et al. 2013)[16]. According to Ghalandar Laki, Negar, et al. (2014), curcumin also has

antitumoral, and antiangiogenesis effects, which simply means that it has tumor inhibiting and

cancer fighting properties. Rightfully, this suggests that it can be a potential preventative for

other diseases, such as cancer, cardiovascular diseases, and macular degeneration (Ghalandar

Laki, Negar, et al. 2014)[7]. An example of its anti-inflammatory effects were shown by a study

conducted by Lim Giselle P., et al. 2001, in which certain doses of curcumin lowered oxidized

proteins and the inflammatory cytokine, interleukin-1, in the brain of APPSw transgenic, or

genetically modified mice (Lim Giselle P., et al. 2001)[13]. In addition to its physical healing

effects, curcumin has been purported to improve cognition. To test this, scientists from Salk

University created the curcumin derived drug, J147, which they orally administered to mice

models of Alzheimers (Prior, Marguerite, et. al 2013)[16]. They found that, when administered in

May 2017
 5

food at a stage when pathology is advanced in 20-month-old transgenic AD mice, rescued the

severe loss of cognitive function, reduced soluble levels of A and increased neurotrophic factors

essential for memory, (Prior Marguerite, et al. 2013)[16]. Specifically, after administrations,

genetically modified mice used to model Alzheimers Disease were able to complete a memory

task almost as well as the healthy mice (Prior Marguerite, et al. 2013)[16]. The scientists attribute

the brain boosting effects to the promotion of the formation of brain-derived neurotrophic factor

by J147, a protein that protects neurons from harmful chemicals, promotes neuronal growth,

connection and the formation of memories (Alzheimers Drug Candidate)[2].

J147s creation was sparked by the need to enhance curcumins neurotrophic effects and

to improve its bioavailability. Purified curcumin has a low bioavailability in part due to its low

water solubility, its rapid metabolism in the body, and the protective layer of cells in the brain

that form whats called the blood brain barrier. Comprised of tight junctions between the

endothelial cells lining blood vessels in the brain, the Blood Brain Barrier can be imagined as the

Great Wall of China of the Brain. Its high efficiency causes it to [prevent] 99% of molecules,

from passing through. (Downs ME, Buch A, et al. 2015)[5]. Purified curcumin, although being

only is 368 Daltons (g/mol), has other molecular properties which cause it to be blocked by the

BBB. Although the BBB protects and surrounds blood vessels, its dysfunction is suggested to be

a symptom of AD. As stated in Banks et al. (2015), A accumulation in the CNS can be both a

cause and consequence of BBB dysfunction in AD, (Banks et al. 2015)[4]. However, the limited

amount of clear cut evidence substantiating the role of BBB disruption in the pathological

progression of AD suggests that other critical functions of the BBB, such as altered transport

and communication within the neurovascular unit, may become impaired in Alzheimers

pathogenesis (Banks et al. 2015)[4].

May 2017
 6

Curcumins bioavailability is further compromised by its low solubility. A review by Lim

Giselle P., et al. (2001) concluded that large amounts of curcumin are expelled by system. It cited

a study that determined that 60-70% of orally administered curcumin was expelled in the feces of

rats, (cited in Lim Giselle P., et al. 2001)[13]. Curcumins bioavailability is further decreased by its

rapid metabolism in the body, its low absorption, and subsequently, its low blood serum

concentrations ("How to Improve Absorption or Bioavailability of Turmeric?")[8]. Focused

Ultrasound Therapy can be implemented to combat this issue and permeate the BBB.

FUS is a technique that uses ultrasound waves to carry energy and disrupt tissue. Using

frequencies between 200-600 kiloHertz, this completely non-invasive technology has great

potential for tumor ablation as well as hemostasis, thrombolysis and targeted drug/gene

delivery, (Kim, Young-sun et al. 2008)[11]. FUS with Microbubbles (MB) has been shown in

many cases to effectively permeate the BBB and facilitate the delivery of several drugs.

Microbubbles are cells containing a gas that injected into the bloodstream . In a pilot study of

BBB opening for cancer-drug delivery to brain tumors by MRI guided FUS, spots that were

treated with FUS and microbubbles were shown by MRI to have clear BBB disruption (Lipsman

2016)[12]. Furthermore, it has been established that FUS and MB can temporarily disrupt the BBB

in targeted regions, allowing therapeutic agents as large as 100 nm in size to the CNS, (Timbie,

Kelsie F., et al. 2015)[21], indicating that the treatment can be implemented to facilitate the

delivery of curcumin as well.

In addition to being a facilitator, FUS can directly target beta-amyloid in the brain. In

studies conducted by Jordo, Jessica F., et. al. (2013) and Leinenga, G., & Gotz, J. (2015), the

application of FUS & MB and Scanning Ultrasound (SUS) & MB, respectively, was found to

result in an increased number of endogenous antibodies attached to the beta-amyloid plaques

May 2017
 7

(Jordo, Jessica F., et. al. 2013; Leinenga, G., & Gotz, J. 2015)[10],[12]. Regarding Scanning

Ultrasound, it is a technique similar to FUS wherein the range of sonication is extended to

throughout the entire brain. Microglia and astrocytes in FUS-treated regions were also activated

and internalized beta-amyloid, although the actual number of cells did not increase, (Jordo,

Jessica F., et. al. 2013; Leinenga, G., & Gotz, J. 2015)[10],[12]. Thus, FUS/SUS & MB itself can

reduce the levels of harmful protein aggregates hypothesized to be initiators of the disease.

Although these activated microglia are strongly believed to be components of inflammation,

Queensland University Alzheimers specialist Juergen Gotz reported that there was no indication

of inflammation caused by these Ultrasound activated microglia in his Scanning Ultrasound

study (J. Gotz, Personal Communication, March 23, 2017). In regards to the study, the subjects

were transgenic AD mice.

An attest to the safety of FUS are the studies conducted by Downs ME, Buch A, et al.

(2015) and Lipsman (2016). In the former, FUS was coupled with microbubbles to disrupt the

BBB in specific regions of the brains of macaques over a 4-20 month period. A pulse length of

10 milliseconds and a total sonication duration of 2 minutes per location resulted in BBB

opening, and BBB closure was observed within a week. A number of tests and MRI scans were

performed and were used determine any effects on cognitive function or any hemorrhage or

edema. Overall, FUS and MB procedure, within given parameters, were shown to be a safe

method to temporarily open the BBB with no long term effects (Downs ME, Buch A, et al. 2015)
[5]
. In the clinical trial by Lipsman (2016), the method utilized to deliver chemo-drugs to brain

tumors by MRIgFUS in humans was with Definity MB ultrasound contrast, a total sonication

time of 50 seconds, with iterations of 2.6 milliseconds on and 30.4 milliseconds off, (Lipsman

2016)[14]. Results showed no severe after effects.

May 2017
 8

From the letter A to Z, there are a few other techniques that can be used in alongside FUS

to optimize the bioavailability of curcumin. One such technique is the application of

nanotechnology. By encasing curcumin in lipid cells similar to microbubbles, the drug can

effectively traverse the BBB due to the properties of the nanoparticles that enhance the cellular

permeability processes of curcumin, (Ghalandar Laki, Negar, et al. 2014)[7]. Nanoparticles such

as liposomes, polymeric nanoparticles, micelles, nanogels, niosomes, cyclodextrins, dendrimers,

silvers, and solid lipids have [also] been shown to deliver therapeutic concentrations of

curcumin, and prove to be extremely useful because they elongate the circulation of curcumin

and provide stronger resistance to metabolic processes, (Ghalandar Laki, Negar, et al. 2014)[7].

Implementing FUS alongside curcumin storing nanoparticles will be much more efficient in

transporting curcumin to the brain than simply implementing FUS with orally administered

curcumin. Nanoparticles will allow for a lower FUS sonication time, thus reducing the temporary

damage done to the BBB.

Piperine, another plant based product, can also improve the bioavailability of curcumin. A

chemical found in black pepper, piperine has been shown to improve the bioavailability of

curcumin by 2000% ( G. Shoba, et al.)[18]. In one human trial, concomitant administration of

piperine 20 mg produced much higher concentrations [of curcumin] from 0.25 to 1 h post drug,

compared to human trials in which 2 grams of curcumin alone were administered and the serum

concentration was nearly undetectable (G. Shoba , et al.)[18]. Thus the pepper based alkaloid can

also be used enhance curcumins bioavailability.

Research Methods and Data Collection:

Since an actual experiment was not feasible nor reasonable, the researcher decided to

conduct a quantitative meta-analysis of five authentic studies. Of these five studies, three

May 2017
 9

analyzed the effects of Ultrasounds on the brains of healthy and/or cognitively impaired animals,

and the remaining two pertained to the effects of curcumin or its derivative on the brain. Charts

were used to record the information in order to compare the findings from each study. The

studies analyzed and respective charts created are Downs ME, Buch A, et al. (2015), Appendix

A; Leinenga, G., & Gotz, J. (2015), Appendix B; Jordo, Jessica F., et. al. (2013), Appendix C;

Prior, Marguerite, et al. (2013), Appendix D; Lim Giselle P., et al. (2001), Appendix E.

Two other studies by Lipsman, Nir. (2016) and Hynynen, K., et. al. (2005) were also

reviewed, but seeing as the former has not been published, and only the abstract of the latter is

available, they were not included in the meta-analysis.

Nine researchers and Alzheimers professionals were contacted with requests for

interviews, but only two responded and only one with agreement for an interview. A Queensland

University Scientist, Professor Juergen Gotz, one of the primary authors of the study analyzed in

Appendix B, was contacted and interviewed over a Skype call on March 23 of this year. A list of

questions asked and paraphrased responses from the interview are found on Appendix H. Other

than clarifying a few misunderstandings about the subject, the interview did not provide the

researcher with essential information.

Results and Data Analysis:

Figure 1, Appendix E illustrates a graph created to compare the differences between

Ultrasound frequencies and pulse repetition frequencies amongst different animal studies. Pulse

Repetition Frequency is the number of pulses of a single frequency that are emitted in one

second. It is dependent on the velocity of sound and on the depth of tissue being interrogated,

(V., Peter J. N.d.)[22]. At a glance, Figure 1 would suggest that smaller animals would require a

higher frequency (or conversely, larger animals would need lower frequencies to penetrate the

May 2017
 10

skull), however, at a careful observation, the little correlation between animal size and frequency

in Figure 1 suggests that the frequency appears to be more of the choice of the experimenters

than requirements for skull penetration, although the FUS/SUS frequencies must be above 200

kHz to be considered Ultrasounds. The same initial hypothesis can be made in regards to Pulse

Repetition Frequency (PRF), but with the little correlation with increasing animal size and

decreasing frequency and the unavailable PRF for the human trials conducted by Lipsman, Nir.

(2016), there seems to be an ambiguous relationship between animal size and PRF as well.

Perhaps the high frequency and PRF used by Leinenga, G., & Gotz, J. (2015) in column 1 of

Figure 1 was due to the use of Scanning Ultrasound as opposed to FUS.

Only the study by Jordo, Jessica F., et. al. (2013) and Lipsman, Nir. (2016) used

Definity as their Microbubble contrast agent, and all of the Ultrasound studies used

microbubbles (Jordo, Jessica F., et. al. 2013; Lipsman, Nir. 2016)[10],[14]. Microbubbles vibrate at

the same frequency of the transducer and can aid in safer BBB disruption.

Although Downs ME, Buch A, et al. (2015) observed BBB closure within a week of

treatment, Professor Gotz stated in his interview that BBB closure should occur within 8-10

hours (Downs ME, Buch A, et al. 2015; J. Gotz, Personal Communication, March 23, 2017)[5].

The one week closing time was probably due to a later measuring date. The BBB closing times

were not available for the other studies, except in the unanalyzed study by Hynynen, K., et. al.

(2005), in which complete BBB closure was observed after 5 hours, with closure beginning

minutes after sonication (Hynynen, K., et. al. 2005)[9]. Therefore, it may be concluded that BBB

reparation begins after sonication and reaches completion within a few hours, and complete

closure time may depend on the method of Ultrasound treatment conducted and the times of

measurement.

May 2017
 11

Microglia and Astrocyte beta-amyloid internalization as a result of Ultrasound treatment

was only studied in Jordo, Jessica F., et. al. (2013) and Leinenga, G., & Gotz, J. (2015). Figure

2, Appendix F is a graph that illustrates the percentage increase in beta-amyloid plaque

internalization by the aforementioned cells after FUS treatment in each study. The beta-amyloid

internalization by microglia in Leinenga, G., & Gotz, J. (2015), which used SUS, increased by

200% after treatment, compared to only 159% in Jordo, Jessica F., et. al. (2013) (Jordo, Jessica

F., et. al. 2013; Leinenga, G., & Gotz, J. 2015)[12],[10]. Astrocyte beta-amyloid internalization was

only measured in the latter and increased by 270% (Jordo, Jessica F., et. al. 2013)[12]. However,

the studies did not specify whether the plaques internalized were soluble or insoluble. Soluble

beta-amyloid is harmful compared to insoluble versions of the same protein. Major treatment-

induced inflammation was not observed in either study. Thus, it can be concluded that FUS and

SUS could inhibit the pathological progression of the cellular aspects of Alzheimers by causing

microglia and astrocytes to internalize beta-amyloid to prevent further accumulation and further

cell death.

The curcumin and derivatives (J147s) studies were conducted by Lim Giselle P., et al.

(2001) and Prior, Marguerite, et al. (2013), respectively. The memory recall of the mice in Prior,

Marguerite, et al. (2013) improved with the administration of J147, as shown in the first column

of Figure 4, Appendix G, in which transgenic mice given J147 were able to locate a target in a

maze 50 seconds before the control mice (Prior, Marguerite, et al. 2013)[16]. Figure 4 also shows

the increase in freezing times for transgenic mice given J147 in the fear conditioning test,

indicating improved performance of the hippocampus and amygdala which are required for

memory formation, memory retrieval, and recall of association to cues (Prior, Marguerite, et al.

2013)[16]. This improvement was hypothesized by the scientists to be a result of increased Brain

May 2017
 12

Derived Neurotrophic Factor protein levels by J147 (Prior, Marguerite, et al. 2013)[16]. The SUS

study conducted by Leinenga, G., & Gotz, J. (2015) saw a minor improvement in mice memory

when the same type of memory recall was tested as in the fear conditioning test (Leinenga, G., &

Gotz, J. 2015; Appendix B)[10]. Mice were taught to avoid a shock zone in a rotating arena, and

the average number of shocks received by mice who had received SUS treatment (and had had

subsequent beta-amyloid reduction) was lower than the sham-treated mice (Leinenga, G., &

Gotz, J. 2015; Appendix B)[10]. Because microglia activation and almost complete beta-amyloid

internalization and minor memory improvement was observed in Leinenga, G., & Gotz, J.

(2015), it indicates that memory could also have been improved by J147 induced beta-amyloid

reduction in the study conducted by Prior, Marguerite, et al. (2013), although there is no mention

of microglia activation in the study (Leinenga, G., & Gotz, J. 2015; Prior, Marguerite, et al.

2013)[10],[16].

Levels of beta-amyloid were reduced in Lim Giselle P., et al. (2001), Figure 5, Appendix

G, by curcumin, and levels of inflammatory and oxidized protein levels were reduced by 61%

(Lim Giselle P., et al. 2001)[13]. In comparison, in Prior, Marguerite, et al. (2013), a 58% decrease

in levels of soluble beta-amyloid and 75% decrease in insoluble beta amyloid in the

hippocampus of untreated transgenic mice compared to transgenic mice given J147 was

observed, as shown in Figure 3, Appendix F (Prior, Marguerite, et al. 2013)[16]. The percentage of

reduction of beta amyloid was not indicated in the first study, neither was the effect on cognition.

Discussion and Conclusion:

Professor Gotz noted that there is no difference between the BBB of mice and that of

humans (J. Gotz, Personal Communication, March 23, 2017). Although it appears that curcumin

might not be able to pass through the BBB of humans, the above note and the positive effects of

May 2017
 13

unaided curcumin administration in mice in the study by Lim Giselle P., et al. (2001) indicate

that curcumin does not seem to be as hindered by the BBB as the researcher previously thought.

However, more evidence is necessary to completely refute the researchers original hypothesis.

FUS/SUS should not be entirely disregarded, for it has been shown to have positive

effects on the brain, with the activation of microglia and subsequent internalization of beta-

amyloid, as well as minute memory improvement with SUS. Curcumin, if altered or

administered with FUS/SUS could also have cognition-improving effects. There might be a

correlation between the reduction of beta-amyloid and improvement in cognition, as shown in

the SUS and J147 study, but other factors, such as increase in levels of BDNF and other

neurotrophic effects of curcumin in the latter study could have come into play with cognition

improvement (Prior, Marguerite, et al. 2013)[16]. Overall, FUS/SUS alongside pure or modified

curcumin shows some promise in inhibiting the pathological progression of Alzhiemers at an

early stage.

Every three seconds, a new case of dementia arises from the depths of the Earth, and its

prevalence is on the rise, with its effects on society set to increase exponentially with the ageing

population (Alzheimers Association)[3]. According to the Alzheimers Association, the number

of people worldwide suffering from dementia is expected to rise from 30 million today, to over

100 million in 2050 (Alzheimers Association)[3]. Alzheimers is the one of the most detrimental

forms of dementia, in which beta-amyloid aggregates and tau protein tangles formed in the brain

are believed to initiate a cascade effect which ultimately leads to paralysis and death. The people

who are affected by Alzheimers branch far off from merely patients; they are intertwined by

neuron-like connections, from caregivers to loved ones, coworkers, friends, and even the

country, which has to pay the expenses for the plights of the disease. The unease arises from the

May 2017
 14

knowledge that these victims die alone in their world, with few memories of their loved ones and

their life. However, the treatment of a component of Alzheimers, inflammation, is believed to be

essential in reducing the pathological progression of the disease. The most potent, natural, anti-

inflammatory agent is curcumin, but its anti-inflammatory properties are futile against the

blockade created by the Blood Brain Barrier. Through the use of FUS, along with microbubbles,

nanoparticles, and even piperine, the bioavailability of curcumin as a cognition improving drug

can be improved and lead to a preventative against Alzheimers.

May 2017
 15

References
[1]
Akiyama, Haruhiko , Steven Barger, et al. "Inflammation and Alzheimer's

disease."Neurobiology of Aging. U.S. National Library of Medicine, n.d.

[2]
"Alzheimer's Drug Candidate May Be First to Prevent Disease Progression - Salk Institute for

Biological Studies." Salk Institute for Biological Studies. N.p., 30 Dec. 2011. Web.
[3]
"Alzheimer's & Dementia Causes, Risk Factors | Research Center| Alzheimer's Association."

Alzheimer's Association. N.p., n.d. Web.

[4]
Banks, William A., et al. "Lipopolysaccharide-induced blood-brain barrier disruption: roles of

cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular

unit." Journal of Neuroinflammation 12.223 (2015): n. pag. Researchgate.net.

[5]
Downs ME, Buch A, et al. (2015) Long-Term Safety of Repeated Blood-Brain Barrier

Opening via Focused Ultrasound with Microbubbles in Non-Human Primates Performing

a Cognitive Task. PLOS ONE 10(6): e0130860. doi: 10.1371/journal.pone.0130860

[6]
Gallagher, Michela. Improving Memory and Brain Function on the Path to Alzheimer's

Disease. Youtube, 13 July 2011. Web.

[7]
Ghalandar Laki, Negar, et al. "Nanotechnology-Applied Curcumin for Different Diseases

Therapy." BioMed Research International. Hindawi Publishing Corporation, 2014.

[8]
"How to Improve Absorption or Bioavailability of Turmeric?" Turmeric for Health! N.p., 10

Apr. 2014. Web.

[9]
Hynynen, K., et. al. (2005). Local and reversible bloodbrain barrier disruption by

noninvasive focused ultrasound at frequencies suitable for trans-skull sonications

[Abstract]. NeuroImage, 24(1), 12-20. doi:10.1016/j.neuroimage.2004.06.046

May 2017
 16

[10]
Jordo, Jessica F., et. al. "Amyloid- Plaque Reduction, Endogenous Antibody Delivery and

Glial Activation by Brain-targeted, Transcranial Focused Ultrasound." Experimental

Neurology. U.S. National Library of Medicine, 2013. Web.

[11 ]
Kim, Young-sun et al. High-Intensity Focused Ultrasound Therapy: An Overview for

Radiologists. Korean Journal of Radiology 9.4 (2008): 291302. PMC. Web.

[12]
Leinenga, G., & Gotz, J. (2015). Scanning ultrasound removes amyloid- and restores memory

in an Alzheimer's disease mouse model. Science Translational Medicine, 7(278).

doi:10.1126/scitranslmed.aaa25127.
[13]
Lim Giselle P., et al. The Curry Spice Curcumin Reduces Oxidative Damage and Amyloid

Pathology in an Alzheimer Transgenic Mouse The Journal of Neuroscience 21(2001):

83708377. Web.
[14]
Lipsman, Nir. "Lipsman - Pilot study of BBB opening for chemo-drug delivery to brain

tumors by MRIgFUS." YouTube. YouTube, 16 Sept. 2016. Web.

[15]
Prince, Martin, et al. World Alzheimer Report 2015. Sept. 2015. England, London.
[16]
Prior, Marguerite, et al. "The neurotrophic compound J147 reverses cognitive impairment in

aged Alzheimer's disease mice." Alzheimer's Research & Therapy. N.p., 14 May 2013.

Web.
[17]
Sahdeo Prasad and Bharat B. Aggarwal. "Turmeric, the Golden Spice." Herbal Medicine:

Biomolecular and Clinical Aspects. 2nd edition. U.S. National Library of Medicine, 01

Jan. 1970. Web.

[18]
G, Shoba, et al. "Influence of piperine on the pharmacokinetics of curcumin in animals and

human volunteers." Planta medica. U.S. National Library of Medicine, n.d. Web.
[19]
Stevens, Michael. Alzheimers and the Brain Online video. Youtube. Youtube, July 22,

May 2017
 17

2016. Web
[20]
Streit, Wolfgang J., et al. "Microglia and neuroinflammation: a pathological perspective."

Journal of Neuroinflammation. N.p., 30 July 2004. Web.

[21]
Timbie, Kelsie F., et al. Drug and Gene Delivery Across the Blood-Brain Barrier with

Focused Ultrasound. Journal of controlled release: official journal of the Controlled

Release Society 219 (2015): 6175. PMC. Web.

[22]
V., Peter J. "Tutorial 1 - Basic physics of ultrasound and the Doppler phenomenon." Tutorial

- Basic physics of ultrasound and the Doppler phenomenon | ICU Sonography. N.p., n.d.
[23]
Weintraub, Karen. "Harvard Researchers Unveil New Alzheimer's Theory." USA Today.

Gannett Satellite Information Network, 2016. Web.

Appendix A

May 2017
 18

Methods/Subject Timeline Details Questions/Links

-Subjects 4 NHP: -Heart rate, blood -Possible edema -Another study
A, N, O, Ob oxygenation, CO2 (atypical has already been
-Adult male, 8-20 expiration, hyperintense conducted by
years, 5-9 kg respiration rate, voxels) only Lipsman et al.
macaques blood pressure found in N & A, (but is not
-Repeated measured after but disappeared published) and
opening of BBB NHP placed in after one week has shown a 220
in caudate & stereotax, 30s b/f -Possible edema kHz transcranial
putamen regions MB injection, 60s had no effects on FUS machine
of Basal Ganglia into sonication, behavioral tests with 2.6ms ON
over period of 4- 30s after -Vital signs (heart sonication time
20 months sonication, and rate, bp, etc.) did and 30.4ms OFF
-500kHz FUS when NHP not change & with a total
transducer removed from heart rate was sonication time
-Acoustic stereotax only greater on of 50s to
Pressures from -MRI scans the first day for effectively and
200-400 kPa; 4-5 acquired 30 min all NHP (possibly safely open BBB
m MB size or 30 hrs after due to unfamiliar in humans
-Pulse length: procedures environment) -How would long
10ms; Pulse -T1 weighted -Reaction time on term memory be
Repetition acquired after test increased for affected?
Frequency: 2Hz; 0.2mL/kg Gad. all NHP on day of -Would different
total sonication administration to procedure, sonication times
duration: 2 min confirm BBB decreased and pressures
per location opening afterward, & and MB sizes be
-NHP -Contrast returned to required for
anesthetized, enhanced T1 baseline 5 days different regions
sedated, hair weighted later of the brain?
removed acquired after 1 -Weight, blood O2 -Would RT and TE
-Catheter IV week & after final levels, resp. change with a
delivered 0.9% procedure to Rate, & bp did larger sample
saline, MB, & MRI verify closure of not significantly size?
contrast agent BBB change, nor did -Stereotactic
gadodiamide -Behavioral tests touch error radiosurgery
-Initial 6s administered to -Histology (SRS) is radiation
sonication w/o test decision unavailable, but therapy that
MB used as making, reaction some petechiae focuses high-
control for time, and and possible power energy on
cavitation accuracy on a damaged a small area
monitoring randomized dot capillaries could Could it be used
-FUS w/MB init. motion target be expected as a instead of FUS?
After bolus test with result of previous -Brain swelling
injection of 2.58 different studies may occur in

May 2017
 19

MB/kg circulated amounts of water -FUS is safe & treatments to the

for 10s droplets as accurately opens brain in SRS
-If caudate & rewards for BBB within given -Effect may take
putamen were different targets parameters weeks or months
sonicated on the -Caudate & -Piezoelectric to be seen
same day, there putamen are transducer -SRS may
was a wait period involved in generates damage tissue
of 20 mins before decision-making electricity when around treatment
restarting MB & and motor pressure changes area
FUS to ensure control, that is
residual MB had why the above
been filtered test was
developed

Downs ME, Buch A, et al. (2015) Long-Term Safety of Repeated Blood-Brain Barrier Opening
via Focused Ultrasound with Microbubbles in Non-Human Primates Performing a Cognitive
Task. PLOS ONE 10(6): e0130860. doi: 10.1371/journal.pone.0130860

May 2017
 20

Appendix B

Methods/Subject Timeline Details Questions/Links

May 2017
 21

-2 cohorts of -After int. APA -Too many things -What was the
male APP23 mice test, mice were were tested in cost of SUS?
(median age, grouped based this study (all not -Equipment?
12.8 months) on performance included) -Procedure?
tested -Received weekly therefore -What does it
-Received SUS or SUS or sham for something is mean if the
sham for 6 weeks 7 weeks bound to be healthy sham
-Beta amyloid -APA retest- SUS positive mice spent
pathology treated mice -Microglia longer with the
measured in were found to number familiar object
brain receive lower avg remained the than the SUS
-2nd cohort of shocks (learned same treated
gender-balanced to avoid zones) -in SUS mice transgenic mice?
APP23 tested than non-SUS models, they -Was it a fluke, or
with healthy mice models, but were activated could the
mice more than sham and contained 2x memory of the
-65 weeks treated healthy more beta- SUS transgenic
median age mice amyloid in mice have
-Memory tested -Novel Object lysosomal improved?
using test conducted to compartments -Why werent
active place test time spent than sham mice other memory
avoidance (APA) with new object models tests
-mice learn to vs. familiar -In cortical areas administered-
avoid shock zone - SUS spent 20 of SUS-treated they could have
in rotating arena, sec with novel mice, Ab tested for
then are tested. and 11 sec was almost different aspects
-Lipid-shelled familiar. completely of memory (as in
microbubbles -Sham spent 18 consumed by Prior Marguerite
with an sec with novel microglial and et al.)?
octafluoropropan and 23 sec with trapped in -Is there any
e core were also familiar lysosomes: seen difference in the
used and mixed -Healthy mice in 75% of the permeability of
in a in a 0.9% with sham spent SUS-treated the BBB of mice
solution of NaCl 19 sec with novel mice; 0% sham and the BBB of
-Concentration of and 15 sec with -Ab-degrading humans?
MB was 1 107 familiar. enzyme IDE -Can the same
to 5 107 -Silver stain (insulin- SUS frequency be
microbubbles/ml showed that SUS degrading translated to
with had 30 beta- enzyme) humans?
size of 1 to 10 amyloid plaques slightly increased - Sonograms
mm and a mean per section vs. by SUS, but no operate at the
diameter of 4 60 plaques in significant same range of
mm. sham mice inflammation frequency as
-Mice models observed SUS, if the
anesthetized with - SUS: 1MHz, -SUS equipment: equipment were

May 2017
 22

Zoletil (20 10-Hz pulse integrated to be modified,

mg/kg) and repetition focused would it be
xylazine (10 frequency, 10% ultrasound possible to use
mg/kg), duty cycle, 6-s system was used sonograms in the
-Head Shaved sonication time (TIPS, Philips Re- place of SUS?
and injected with per spot. search) (16): -How long did the
1 ml/g MB -1.5 mm between annular array BBB remain
solution multiple transducer with open?
- Put under sonication natural focus 80 - At what stage in
ultrasound sites so that mm, radius of the disease
transducer & ultrasound was curvature 80 would
treatment delivered to the mm, spherical
SUS be most
delivered (none entire brain shell
for sham) 80 mm, central effective?
opening 31 mm
diameter, 3D
positioning
system, &
programmable
motorized
system to move
the ultrasound

- No difference of
NF-kB in SUS and
sham transgenic
mice
-SUS showed no
DAMAGING
inflammation

May 2017
 23

Leinenga, G., & Gotz, J. (2015). Scanning ultrasound removes amyloid- and restores memory in
an Alzheimer's disease mouse model. Science Translational Medicine, 7(278).
doi:10.1126/scitranslmed.aaa25127.

Appendix C

Methods/Subject Timeline Details Questions/Links

May 2017
 24

-20 4 month-old Fus-0.3 MPa, -How long did the

-TgCRND8
male and female 120 sec total, BBB remain
MRIgFUS treated
non-Tg and 10 ms bursts, 1 open?
mice- 4 day
TgCRND8 mice Hz repetition -What was the
sacrifice (post-
each rate effect on
treatment) After
-Mice 0.5MHz cognition? (if any)
one treatment,
anaesthetized frequency
plaque size -Conducted in
with 150 mg/kg transducer
decreased by Sunnybrook
ketamine and 10 - 4 targets, 1.5
20% and A-Beta research
mg/kg xylazine mm apart,
surface area by institute- same as
-Placed on a small -Positioning
13% (compared Nir Lipsman
animal MRIgFUS system moved
to corresponding -What is the
-Separate cohort into 1.5T MRI
untreated region significance of
used for scanner
on the left side) the activated
immunohistochem -Treated only
-4 hours after microglia/astrocyt
istry analyses: one hemisphere
treatment, es being close to
sacrificed at 4 of the brain
microglia or far from
hours, 4 days, and -T2- and T1-
expressed plaques?
15 days. 6 of 9 weighted
protein Iba1
TgCRND8 mice imaging
increased 2.8 &
used at 4 days - BBB opening
2.9-fold
post-MRIgFUS had determined with
compared to
previously been MRI gadolinium-
untreated
an MRIgFUS based contrast
corresponding
group- 3 mice agent (only
regions.
added enters if BBB
endogenous
-MRI gadolinium made more
-4 days post
contrast agent permeable)
treatment IgG
and FUS -After
(immunoglobulin
microbubble treatment,
G antibody) and
contrast agent second cohorts
IgM (M antibody)
(Definity) brains were
were bound to
administered b/f removed, then
plaques in
treatment saturated in
treated region
30% sucrose
-Untreated
solution. - 40
cortex, plaques
m cut coronal
only had trace
sections
amounts of
-Microglia mouse IgG and
stained with no mouse IgM
rabbit anti-Iba1 -No increase in
(1:1000; Wako microglia cell
Chemicals) number
-Astrocytes -Microglia far from

May 2017
 25

stained with plaques internalized

goat anti-GFAP 159% more A
(1:500; AbD counts in treated vs
Serotec) untreated cortex
-Microglia close to
-A surface plaques internalized
area: estimated 52% more A in
total number of treated vs untreated
plaques cortex
multiplied by -Astrocytes far from
mean plaque plaques internalized
size 270% more A in
treated vs untreated

Jordo, Jessica F., et. al. "Amyloid- Plaque Reduction, Endogenous Antibody Delivery and
Glial Activation by Brain-targeted, Transcranial Focused Ultrasound." Experimental Neurology.
U.S. National Library of Medicine, 2013.

May 2017
 26

Appendix D

Methods/Subject Timeline Details Questions/Links

May 2017
 27

-Male & Female Water Maze: Improper APP Would J147 work
Transgenic AD- 100sec cleavage is in humans?
APPswe/Pi^E9 escape time caused by beta How would a
mice; 20 months AD + J147- ~50 secretase, whose
J147+don. Work
old sec level was
-Test group fed Scopolamine + reduced by J147, in transgenic
high fat diet and Healthy mice- thus increasing mice?
10mg/kg/day J147+don. And levels of whole What is the cost
J147 for 3 J147 halved the APP. efficiency of
months. time APP may have a synthesizing
Cognition tests: Elevated Plus role in synaptic J147?
-Water maze Maze: structure and
of donepezil?
used: trained AD- ~15 sec time function as well.
mice to find a in open arms In the Are there any
platform within AD + J147- ~1 scopolamine side effects of
180sec, some sec trials, the J147 + either drug?
could not do (implies a better donepezil combo. Can
within time limit, anxiety improved times J147+donepezil
then J147 response) the most for all permeate the
administered Fear Conditioning tests.
BBB?
mice took the Day 1: BDNF & NGF
test -Elevated AD- ~10 sec time stimulate growth Answers:
plus maze used: frozen and maintenance J147 is $89 for
maze with 2 AD + J147: ~20 of neurons, 2mg (solid) and
open & 2 closed sec survival of is 350 Daltons
arms used to Scopolamine + mature neurons, Donepezil:
measure mices Healthy mice- and play a role in
100%
response to J147 + donepezil synaptic
anxiety. Mice had the longest plasticity, bioavailable,
were habituated time frozen after cognition, and category c
to the room control group memory pregnancy risk,
25hrs b/f & Fear Cond. Day formation. Levels 379.5g/mol, $70
habituated to the 3: of these increase for 10mg, $110
maze 2min b/f AD- ~2 sec time in response to for 100mg
the test. frozen neuronal injury.
-Fear AD + J147- ~3.5 -Fear
conditioning: Day sec conditioning:
1- mice explored Scopolamine + measures ability
a chamber for 2 Healthy mice- of hippocampus
min and J147 alone, & amygdala to
conditioned to donepezil alone form and receive
fear tone with a and J147 + don. new memories:
30sec tone Increased
followed by 2sec freezing time,
foot shock. but combo did

May 2017
 28

Repeated after the most

30 sec. and
exploration J147 decreased
resumed for 2 soluble (harmful)
min & insoluble levels
Day 2: Mice of Amyloid Beta
explored as well as Beta
chamber but secretase
without tone and Increased levels
shock of APP, pro &
Day 3: Same mature Nerve
procedure as day Growth factor
1 but w/o shock. and BDNF levels,
A camera and decreased
measured pro to mature
freezing time for NGF & BDNF
days 1 and 3. ratios.
Second test: Increased target
Scopolamine gene levels of
induced memory BDNF: Homer 1 &
impairment 30 Erg 3.
min b/f testing on Homer 1: Helps
healthy mice who in signal
were fed J147 or transduction,
J147 + donepezil, synaptogenesis,
only donepezil, and receptor
or saline (for trafficking at
control) All tests, synapses
plus Y-maze, Erg 3: Helps in
were performed synaptic
plasticity,
learning and
memory and
processing Short
& Long term
hippocampal
dependent
memory

Prior, Marguerite, et al. "The neurotrophic compound J147 reverses cognitive impairment in
aged Alzheimer's disease mice." Alzheimer's Research & Therapy. N.p., 14 May 2013.

May 2017
 29

Appendix E

Methods/Subject Timeline Details Questions/Links

May 2017
 30

-Ten-month-old -Mice injected -Limitation of Info from the

male and female with 0.9% normal using NSAID for intro:
APPSw Tg + saline, followed AD prevention of - Regions using
(transgenic) and by HEPES buffer, AD is NSAIDS have a
Tg (healthy) pH 7.2, gastrointestinal lower risk for
mice from 12 containing 5 toxicity caused Alzheimers
litters mg/ml each by inhibiting (Breitner et al.,
-Randomly leupeptin and cyclooxygenase I, 1995; Stewart
grouped aprotinin and 2 which could be et al., 1997).
- For 6 months, mg/ml pepstatin overcome using -Regular
Tg+ mice were -Single curcumin ibuprofen
fed 1) chow with hemisphere brain -Tg+ untreated treatment
low dose of regions dissected mice had 55 significantly
curcumin (160 using mouse nanograms of suppressed
ppm)), or 2) a brain atlas beta-amyloid in inflammation and
high dose of coordinates. treated region the development
curcumin (5000 -Thalamic, -Tg + with low of -amyloid in
ppm), or 3) no cortical, and dose of curcumin Tg2576 APPSw
drug hippocampal had 33 transgenic AD
- Tg fed fed regions, and nanograms of mice (Lim et
chow without entorhinal cortex beta-amyloid in al., 2000).
-ELISA used to and piriform treated region - The above links
measure beta- cortexamygdala -High doses of are some studies
amyloid sections curcumin, referenced in the
-Oxidative dissected out showed no analyzed study
damage and snap frozen change in the that are worth
measured in in liquid nitrogen. amount of looking at
residual cortex -Biochemical insoluble A in _________________
and piriform measurements the brains of ___
cortex of Tg + taken in the treated mice -Why didnt high
mice. hippocampus, -Levels of doses of
-At sacrifice time, entorhinal cortex, inflammatory curcumin work?
there was no piriform cortex cytokine -Can ibuprofen
significant amygdala, and interleukin B be used instead
change in weight residual cortex were reduced by of curcumin?
and no indicator -Tissue samples 61.8% in Tg+ -Does this mean
of diet-related were with curcumin. that curcumins
toxicity homogenized in -Levels of bioavailability is
10 vol of TBS oxidized proteins normal?
containing many reduced by -Did the
protease 61.5% cognition of the
inhibitors (20 -PT-stained area mice improve at
g/ml each on cryostat all?
pepstatin A, sections in Tg- -Why are soluble
aprotinin, and TG+ were beta-amyloid

May 2017
 31

phosphoramidon, used to measure plaques the

and leupeptin, microglia target of these
0.5 mm PMSF, activation studies (vs.
and 1 mm EGTA). -Two-way ANOVA insoluble)?
-Tissue sonicated showed that -Cyclooxygenase-
briefly (2xfor 10 percentage of PT- 1 is an
sec) and stained area was inflammatory
centrifuged at reduced by 31% mediator, why
100,000 g for -Inner plaque would its
20 min at 4C. staining was not inhibition not be
reduced by seen as
curcumin, but beneficial?
was increased -Wouldnt
directly outside curcumin inhibit
of the plaques it?

Lim Giselle P., et al. The Curry Spice Curcumin Reduces Oxidative Damage and Amyloid
Pathology in an Alzheimer Transgenic Mouse The Journal of Neuroscience 21(2001): 8370
8377.

May 2017
 32

Figure 1
Frequency and PRF Comparisons

Appendix F

Figure 2

May 2017
 33

Percent Increase in Beta-Amyloid Internalization by Activated Microglia and Astrocytes after

Ultrasound Treatment

Figure 3
From Prior Marguerite, et al. 2013

Appendix G

May 2017
 34

Figure 4
From Prior Marguerite, et al. 2013

Figure 5
From Lim Giselle P., et al. (2001)

May 2017
 35

Appendix H

1. Is there any difference in the permeability of the BBB of mice and the BBB of
humans?
a. No, but sheep do have a different BBB than humans.
2. What was the cost of the scanning ultrasound used in your study?
a. About $80,000.
3. What is the average cost of FUS?
a. About $150k-200k including equipment.
4. Can the same SUS frequency be translated to humans?
a. A 1Mz frequency was used in the study. Humans have a thicker
skull, therefore a lower frequency would be required to penetrate it.
5. Sonograms operate at the same range of frequency as SUS, if the equipment
were to be modified, would it be possible to use sonograms in the place of SUS?
If yes, what are possible modifications?
If no, please elaborate
a. Sonogram frequencies are higher than those of FUS/SUS. If the
steering mechanism was modified and a lower frequency was used, it could be
used as an alternative for FUS/SUS, but again, he is not an ultrasound expert,
but an Alzheimers expert.
6. How long did the BBB remain open?
a. About 8-10 hours.
7. At what stage in the disease would SUS be most effective?
a. Vague answer: Most likely in the early stages.
8. Would it be used as a preventative or a treatment?
a. Most likely as a treatment.
9. Could SUS be used repeatedly in humans without extensive brain damage?
a. Yes, it is safe when conducted in the right parameters.

May 2017