COMPENSATORY CORTICAL SPROUTING

ACROSS THE LIFESPAN OF THE RAT

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A Thesis

Presented to

The Honors Tutorial College

Ohio University

______

In Partial Fulfillment

of the Requirements for Graduation

from the Honors Tutorial College

with the degree of

Bachelor of Science in Biological Sciences

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by

Benjamin J. Carnes

April 2016

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This thesis has been approved by

The Honors Tutorial College and the Department of Biological Sciences

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Dr. Sonsoles de Lacalle

Professor, Biomedical Sciences

Thesis Advisor

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Dr. Soichi Tanda

Honors Tutorial College, DOS

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Jeremy Webster

Dean, Honors Tutorial College Carnes 3

TABLE OF CONTENTS

Acknowledgements…………………………………………………………………….5

Abstract………………………………………………………………………………...6

Introduction...... ……………...…………………………………………………....….8

The Importance of Alzheimer’s Disease…………………….………....…….....8

Alzheimer ’s Disease and the Cholinergic System.…….….…………...………9

Neuropathology of Alzheimer’s Disease………………….……………………9

AD Genetics...…………….....…………………………….…………….……..11

Epidemiology…………………………………………….…………………….13

The Relationship Between the Cholinergic System and Dementia..…………..14

Current Therapies for AD...... ……………………………...16

Basal Forebrain Cholinergic System...... ………………………………17

Connections of the EC and HDB...... …………………….…………19

Compensatory Mechanisms...... ………………………………21

Potential Therapies for AD...... ………………………………23

Compensatory Cholinergic Sprouting: An Analysis of Feasibility...... …...... 24

The Question...... …………………….…....….…24

Hypothesis...... ……………………...... ……25

Overview and Rationale...... …………………….....………25

Rat Models and Aging Effects...... ………………….……....……26

Experimental Induction of Cell Loss...... …………………….....………27

Description of 192-IgG Saporin...... …....…………………….……28 Carnes 4

Rational for the Location of the Lesion...... ………………………………29

Rationale for the Choice of Histochemical Technique...... …………29

Rationale for the Choice of Image Analysis Technique...... ……31

Data Analysis Tools...... ………………………………34

Significance...... ………………………………34

Methods……………………………………………………………………………….36

Subjects...... ……………………………………….………………….36

Administration of the Immunotoxic Lesion...... ……………………………36

Tissue Collection..………………………………….………………………….37

Staining.…………………………………………….………………………….37

Data Analysis.…………………………………….……………………………41

Statistics...... ………………………….…………………………………44

Results………………………………………………………………………………...45

Effect of the Toxin on the HDB....…………………………………………...... 45

Effect of the Lesion in the HDB on the EC....……………....…………………46

Discussion...... …………………………………………....…………….………….53

Future Directions...... …….....……………………………………………….....57

References…………………………………………………………………………….59

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Acknowledgements:

I would like to start by thanking my thesis advisor, Dr. Sonsoles de Lacalle, for her editing and constant support throughout this process. I am grateful to Dr. Soichi

Tanda for his support and guidance. My thanks to Assistant Dean Cary Frith, Dean

Webster, the HTC faculty, and all faculty I worked with over the past four years who have given me opportunities and helped me become a better student and person.

Finally, I would like to thank my friends and family for their constant encouragement, positivity, and support.

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Abstract:

To investigate the plastic capacity of the cholinergic system in a partial animal model of Alzheimer’s disease, adult and aged rats received unilateral lesions of the horizontal diagonal band of Broca (HDB) using the cholinergic-specific toxin 192-

IgG-saporin. The contralateral HDB in the rat was used as a control. The rats were sacrificed at 2, 4, 8, 12, or 24 weeks post lesion. Immuno- and histochemical techniques were used to quantify the effects of the lesion. Tissues were stained using an acetylcholinesterase technique. A 230µm by 200µm grid was used to indirectly measure the density of cholinergic fibers in the Entorhinal Cortex (EC).

All groups (young: 3; young adult: 12-15; adult: 18; aged: 24-27 month old rats at the start of the experiment) exhibited a decrease in cortical fiber density after the lesion, which was more pronounced in the young group. As the rat ages, we discovered that there is a decreased sprouting response. In 3 mo rats (young), there was an increase in fiber density from 2 weeks to 24 weeks post lesion of 10.8%. In the

12-15 mo (adult) group, there was an increase of 21.5% between 4 weeks and 12 weeks post lesion. However, in 12-15, 18-20, and ≥24 mo rats, there was no cholinergic fiber recovery past the 2 weeks post-lesion fiber density.

From the obtained results, we speculate that in 3 mo and 12-15 mo rats, following a cholinergic specific lesion, there is at least to some extent a compensatory mechanism activated in the basal forebrain such that surviving neurons, projecting to the same target, are able to extend terminals and occupy the denervated area. It Carnes 7

remains to be investigated whether the sprouts are able to establish proper synaptic connections and make a functional recovery.

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INTRODUCTION

1. The Importance of Alzheimer’s Disease:

An estimated 5.2 million Americans are affected with the neurodegenerative disorder known as Alzheimer’s disease (AD). This number is expected to reach 13.8 million by 2050 (Association 2014). AD is a form of dementia, which is a broad term for diseases characterized by a loss of memory or other skills required for everyday life. All types of dementia are ultimately caused by damage to nerve cells in the brain.

The death of these cells, called neurons, is responsible for the change in memory and behavior seen in dementia. Depending on its causes and areas of the brain affected, different types of dementia exhibit different cognitive changes; each type can be identified by its symptoms and treated differently. Around 60-80% of all dementia cases are AD (Association 2014). This chronic neurodegenerative disease often begins with short term memory loss, and as it progresses symptoms can include language issues, disorientation, and behavioral changes. There are many risk factors, and the more commonly cited include an age of over 65, family history, mild cognitive impairment, and cardiovascular disease (Association 2014). Recent studies are showing that social engagement and more formal education can lead to a decreased risk for AD (Galimberti and Scarpini 2014). While many clinical characteristics have been identified, AD is an important area of research because we do not fully understand its cause and thus have no cure.

My thesis will approach AD knowing that the cause and mechanism for the disease are unknown. Instead of searching for the causes of this disease, my lab Carnes 9

attempted to find a way to stall the symptoms of AD. Our approach was targeted at helping those who have AD, rather than preventing it. Before I discuss my work, I want to share where my research fits in to a bigger picture. There are many levels of research that lead to clinical advancements. Therapeutics cannot be approved until clinical trials are completed. Human trials cannot start until there have been successful animal studies showing improvement. These animal studies cannot start without some understanding of expected outcomes of whatever drug will be used or an area of the body or physiological process in the body to target. This is where my thesis work comes in. I attempted to elucidate a target, specifically cholinergic neural sprouting, which could be upregulated, slowing progression and improving life for AD patients.

To better understand why we examined sprouting, I will describe what is known of

AD, important areas and functions of the brain, and previous work in the field.

2. Alzheimer’s Disease and the Cholinergic System

2.1 Neuropathology of Alzheimer’s Disease:

In 1906, Dr. Alois Alzheimer presented for the first time a clinical case of early-onset dementia and the corresponding neuropathological findings. By 1911, his description of the disease was being used by physicians to diagnose patients.

Neuropathological stages of AD have been refined throughout the years (Association

2014; Serrano-Pozo and Frosch 2011), but the pathological cellular hallmarks always include amyloid plaques and neurofibrillary tangles (Fig. 1). Carnes 10

Amyloid plaques are misfolded protein aggregates that build up in the brain of aging individuals and AD patients. Researchers have debated for years whether these plaques are the causative agent in AD. Proponents of the amyloid plaque hypothesis of

AD are examining biological processes that occur just before plaque formation, such as proteolytic processing (Espeseth et al. 2005). No mechanism or working treatment has been found, but amyloid plaque formation in AD is clearly involved, making it a central area of research.

Fig. 1 – Alzheimer’s Disease – Cellular and Morphological Characteristics

The diagram depicts a cartoon of diseased neurons and the disintegration of microtubules leading to cell death. Coronal sections of the brain show morphological changes in AD (Morreale 2009).

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Neurofibrillary tangles are hyperphosphorylated tau proteins that accumulate inside the neurons, causing cell death (Braak and Braak 1991). Tau proteins normally help stabilize microtubules, which provide a path for movement of substances throughout the neuron. These tangles of improperly functioning tau lead to degradation of microtubules, destroying the cytoskeleton of neurons and leading to cell death. Over the years, researchers in this field have identified a correlation between the clinical symptoms of dementia and the extent of neuropathological damage, and this work has also helped to establish a topographical chronology in the progression of AD. For example, amyloid plaque build-up often begins before cognitive deficits, while neurofibrillary tangles, neuron loss, and synaptic loss progress with cognitive decline (Serrano-Pozo and Frosch 2011). Although these neurofibrillary tangles correlate well with neurological damage, the earlier appearance of amyloid plaques in AD has led some to believe that tau is not the primary cause of

AD. Nonetheless, continuing research in this area is essential to halt the progression of neurofibrillary tangles and the development of plaques.

2.2 AD Genetics

Currently, there are two classifications for AD: early onset and late onset.

Early onset AD is quite rare, believed to be occurring in only 5% of all AD cases.

Familial AD (FAD) makes up most of these cases, and usually presents between the ages of 30 and 60, due to a mutation in at least 1 of 3 possible genes. These mutations are located on different chromosomes, which include 1, 14, and 21 (National Institute Carnes 12

on Aging 2015). Although these cases of AD make up such a small percentage of the total, they have helped elucidate information on the disease, and have given researchers clues to how AD begins.

On chromosome 21, the mutation in the gene encoding amyloid precursor protein (APP) leads to FAD. When this mutation occurs, the abnormal protein will be improperly processed and result in formation of the toxic amyloid beta peptide. On chromosomes 14 and 1, mutations in the Presenilin 1 and 2 genes, respectively, have been indentified. These genes code for proteins that help enzymatically cleave APP, forming the amyloid beta peptide (Ertekin-Taner 2007). Mutations in these three genes eventually cause amyloid plaques to form. These findings have helped scientists better understand all cases of AD and the progression of the disease. Based on these studies, we now know important steps in amyloid plaque formation, and other proteins that play a role in AD.

Late onset AD, also known as sporadic AD, is much more complicated and makes up about 95% of AD cases. Genetic factors are involved, but a majority of these factors are poorly understood. The apolipoprotein E (APOE) gene, found on chromosome 19, is the only well-known genetic risk factor. This protein helps carry cholesterol through the blood, and can also help carry cholesterol to neurons. There are several forms (alleles) of this gene, APOEε2, APOEε3, and APOEε4. Everyone has two alleles of the gene, as we have two of each type of chromosome. These alleles differ in several amino acids, leading to different numbers of cysteine residues per mole (Rall, Weisgraber, and Mahley 1982). APOEε2 is a relatively rare allele, and Carnes 13

may have some protective influence against AD. This version of apolipoprotein E does not bind well to cell surface receptors, but it is unknown what the exact molecular mechanism is that influences AD. APOEε3 is the most common allele and could be considered the “wild type” allele. It has been shown to have no influence on the development of AD. APOEε4 on the other hand, increases the risk of AD. With two copies of APOEε4, the risk for AD is greatly increased (National Institute on Aging

2015). The risk factor for AD is not categorized as dominant or recessive. Depending on the combination of alleles, the risk factor can be increased and decreased to different increments (Farrer et al. 1997). While this one genetic risk factor is important, it is still not fully understood how it influences AD. Sporadic AD is currently viewed as a combination of genetic, environmental, and lifestyle influences, all of which combine to create a confusing and yet to be elucidated causal mechanism for AD.

2.3 Epidemiology

Dementia in the US is predicted to increase in prevalence over the coming years, and this is also true in other countries. Dementia effects 46.8 million people worldwide, with global care costing an estimated 818 billion dollars. The number of cases is expected to increase by 2030 to 74.7 million, and over 130 million by 2050, costing trillions of dollars. A trend has been found showing an increase in dementia cases for low and middle class people, and the need for research and treatment will only increase in poor and underserved regions of the world. (World Alzheimer Report Carnes 14

2015) Thus, the importance of research in the field cannot be stressed enough, as a breakthrough in genetics, neuropathology, and possible treatments can improve or save millions of lives.

2.4 The Relationship Between the Cholinergic System and Dementia

The discovery of chemical transmission of signals in the brain, specifically the neurotransmitter acetylcholine (ACh) was awarded the Nobel Prize in 1936. From the

1960’s to the 1980’s, many of the most important neurotransmitters were discovered and many neuroanatomical pathways were delineated in many different publications that examined everything from biochemistry to morphology. These discoveries provided a better understanding of ACh and the basal forebrain cholinergic system of neurons. Studies of geriatric and AD brain tissue from the basal forebrain demonstrated a decrease in enzymes used to synthesize ACh, as well as a decrease in the reuptake of ACh (Contestabile 2011). These results contributed to establish the proposed link between memory, cholinergic neurons, and aging.

Earlier, in 1982, a landmark paper by Bartus and colleagues published in

Science had proposed the “cholinergic hypothesis of geriatric memory dysfunction”, which provided an extensive explanation for how deficits in cholinergic neurotransmission underlies cognitive decline in aging. Later work confirmed this finding in rats (Luine and Hearns 1990) and the relationship of the cholinergic system with memory decline, and these relationships have been extensively investigated in many publications since. By extension, this cholinergic hypothesis also applies in the Carnes 15

case of AD, because it had already been shown that in AD there is a substantial loss of cholinergic neurons in the nucleus basalis of Meynert (Whitehouse, Price, and Clark

1981). The nucleus basalis of Meynert, in the basal forebrain (Fig. 3), contains a subset of magnocellular cholinergic neurons, identified by the presence of the synthesizing enzyme choline acetyltransferase (ChAT). In the last fifty years, a large body of evidence has documented that cholinergic basal forebrain neurons provide the main cholinergic input to the whole cortical mantle in many species, including humans

(Woolf 1991). The involvement of cholinergic neurons in AD has also been clearly documented by the scientific community, as mentioned above (Cuello et al. 2009;

Mufson et al. 2008; Kása, Rakonczay, and Gulya 1997; Iraizoz et al. 1999). In fact, a study that injected ACh-secreting cells into the cortex of memory impaired aged rats demonstrated an improvement in cognition (Dickinson-Anson et al. 2003). This further supports the importance of ACh and the cholinergic system to memory and aging.

The cholinergic hypothesis of AD has gained traction in recent years as the possible primary cause of AD’s cognitive decline (Shen 2004). In fact, a recent study examined preclinical cases of AD and the response of the brain when a low dose of temporary cholinergic neurotransmitter disruptor was administered to examine otherwise undetectable cholinergic deficits. Unlike the control patients, all those with preclinical AD were found to have significant issues with cognition at 5 hours post- dose (Lim et al. 2015). This implies that cholinergic system deficits occur in the onset of AD. While there is still a large amount of work to do to discover the mechanism of Carnes 16

AD, what cannot be disputed is that the basal forebrain cholinergic system is important to memory and the progression of AD.

2.5 Current Therapies for AD:

Without a known mechanism of AD to help guide research for a cure, current therapies can only slow the progression of the disease. These treatments relate to an

Fig. 2 – Representation of Acetylcholinesterase Inhibitors in Cholinergic Synapses

The diagram shows the synapse between two neurons. The pre-synaptic neuron on top is a cholinergic neuron, which releases acetylcholine. Cholinesterase inhibitors block the breakdown of acetylcholine, allowing acetylcholine to be present for a longer period of time. In AD, this creates a stronger signal, slowing the progression of mental disturbances (Bondre 2015).

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aspect of the cholinergic system. In 1993, after years of research and trials, the first acetylcholinesterase (AChE) enzyme inhibitor was released as a treatment of symptoms for AD (Ibach and Haen 2004). The original version of the drug is no longer prescribed due to its liver toxicity, but there are three FDA approved versions currently in use. The AChE inhibitor molecules modify the action of the AChE enzyme, which normally breaks down ACh in the brain, allowing ACh to remain active longer to compensate for reduced levels following the loss of cholinergic neurons (Fig. 2). Treatment with AChE inhibitors reduces behavioral disturbances, enhances memory in the short term, and slows the progression of the disease. (Auld,

Kornecook, and Bastianetto 2002) As the disease progresses, possibly because the number of surviving cholinergic neurons becomes insufficient, the treatment with inhibitors is no longer as effective. This failure of palliative treatment shows the importance of finding a better approach, but to do so we need to better understand the neurobiology of the basal forebrain and the mechanisms by which those neurons degenerate. My work, as discussed later, will contribute to this by identifying the recovery capability of the basal forebrain cholinergic neurons (BFCN) in response to denervation over time.

2.6 Basal Forebrain Cholinergic System:

It is important to understand what BFCN are. A “cholinergic” neuron is one that produces the neurotransmitter ACh. Neurotransmitters send signals to other Carnes 18

Fig. 3 – Representation of Cholinergic Pathways in the Rat

The diagram depicts sources and pathways of cholinergic input to areas of the brain in the sagittal plane. On the left is the rostral side of the brain, on the right is the caudal side (Woolf 1991). neurons, triggering responses in other areas of the brain that are manifested in outputs such as movement, behavior, memory, etc. ACh is found in many areas of the brain as well as in neuromuscular junctions.

Anatomically, BFCN form a continuum extending rostrally from the medial septum (MS) and horizontal and vertical diagonal band of Broca (HDB, VDB), through the substantia innominate (SI), to include the nucleus basalis (Bas) caudally

(Fig. 3). Functionally, these BFCN provide a topographically organized innervation to the entire cortex (Cuello and Sofroniew 1984; Saper 1984; Sofroniew, Pearson, and

Powell 1987). This means the axons of these cholinergic neurons extend throughout the cortex (as shown in Fig. 3) where they use ACh to communicate with other neurons. This has been confirmed, as lesions of the BFCN lead to a decrease in levels Carnes 19

of ACh in the cortex (Coyle, Price, and DeLong 1983; Braak and Braak 1991). The areas the BFCN project to include the Entorhinal Cortex (EC) (Fig. 4), an area of the cortex located in the temporal lobe, known to be linked to memory after the lobotomy of Henry Moliason in the 1950’s (Scoville and Milner 2000). Interestingly, the EC is the cortical region that shows signs of degeneration (accumulation of plaques and the buildup of neurofibrillary tangles, as well as cell loss) in the early stages of AD (Khan et al. 2014). Because of this early involvement in AD, I am focusing my work on the

EC and cholinergic sprouting.

2.7 Connections of the EC and HDB:

Using track-tracing techniques and staining specific to cholinergic neurons, connections to and from the EC were found in parts of the MS and VDB, as well as connections to other areas (Fig. 3). These areas include the hippocampus, which is also important for memory, and the mesolimbic cortex, a rostral area of the cortex

(Fig. 3), which is important in reward behavior (Woolf 1991). The EC itself sends projections to the MS and VDB, as well as the hippocampus. In regards to my research, this network of connections make it hard to isolate the EC and study the effects of a lesion of the BFCN on the EC without denervating other areas of the brain that connect to the EC. If the MS or VDB is lesioned, it will denervate part of the EC, but it will also lead to denervation in the hippocampus and areas the EC projects to.

This means denervation will be present on the efferent and afferent neuron Carnes 20

Fig. 4 - Connection between the HDB and EC

Pictured is a diagram of a horizontal section through the rat brain. The red line shows the cholinergic neural pathway extending from the HDB and innervating the EC (Paxinos and Watson 1998). connections of the EC, which would introduce more variables and possibly cause even more cell death.

The lateral and intermediate sections of the HDB have efferent connections to the lateral EC and the olfactory nuclei (Gaykema et al. 1990). This means that if neurons die in the HDB, both the EC and the olfactory cortex will be denervated.

However, the loss of some neurons and axon fibers in the olfactory nuclei pathway does not affect axon fibers in the EC. This makes the HDB a good target for a lesion Carnes 21

that will denervate the EC without causing denervation or cell death in other areas of the brain that connect with or affect the EC.

We also chose to target the HDB cell bodies in our study of the EC because those neurons send axons to the EC of the same side, without contralateral projections

(Fig. 4). The lateral HDB also has few other connections that would be effected by a lesion of its cell bodies. Damaging many different areas of the rat brain could lead to unexpected variables and large amounts of undesirable cell death. Therefore, the cholinergic projection from the HDB to the EC was chosen as the focus of my study in rats (Fig. 4) as the connection does not induce other variables to the study of the EC.

2.8 Compensatory Mechanisms:

There are compensatory mechanisms in the nervous system that address deterioration due to disease or aging. A recent review revealed that the brain may reorganize functionally to compensate for age related deterioration (Morcom and

Johnson 2015). Another compensatory mechanism, sprouting, is a well-known occurrence, with one of the earliest studies on the topic occurring in the 1950’s (Liu and Chambers 1958). The authors found that after partial denervation of the cat spinal cord, the axons of surviving neurons re-innervated areas that were once occupied by the axons of dead neurons. More recent studies have shown lesion-induced sprouting in different areas of the brain including the primary and somatosensory cortex and the hippocampus (van Groen, Miettinen, and Kadish 2011; Szigeti et al. 2013). The Carnes 22

Fig. 5 – Diagrammatic representation of axonal sprouting following basal forebrain cholinergic neuron lesions. (Healthy neurons in red, damaged neurons in green)

The diagram shows normal cholinergic cells with their axons innervating the cortex (represented by the rectangle) in A. When a cholinergic neuron dies (B), it leaves a dennervated area in the cortex. Compensatory axonal sprouting (C) from surviving cells can occur, which will re-innervate the area (Hartonian and de Lacalle 2005).

concept is that when neurons die, their axons wither away, leaving an area denervated.

The axons of surviving neurons will sprout, re-innervating the area (Fig. 5).

The brain contains many neurons outside of the cholinergic pathway, so it is important to understand how selective the axonal sprouting response is. This was shown in several studies using histochemical staining of the brain and spinal cord after a lesion. The authors demonstrated that neurons in the same pathway of the same type, such as cholinergic neurons, will sprout and re-innervate areas that were innervated by other cholinergic neurons. This finding confirms that the axonal sprouting response is selective and restricted to the axon fiber population already present in the area (Kuang and Kalil 1990; Ramirez 2001). The molecular mechanism of sprouting requires Carnes 23

further research, but it is speculated that this outgrowth has to do with local signals, similar to those in development (Kuang and Kalil 1990). Finding these signals is an important area of research in the field. However, I examined whether the sprouting caused by these signals occurs in the first place, specifically in the cholinergic system over the lifespan of the rat.

2.9 Potential Therapies for AD

Increasing ACh using a sustainable method would be a major step in treating those with AD, a large improvement over the current pharmacological AChE inhibitors. An interesting study mentioned earlier examined implants of ACh-secreting cells in the cortex of aging rats. The researchers determined, through a series of behavioral tests, that these implants improved age related cognitive impairments in rats (Dickinson-Anson et al. 2003). Unfortunately, injecting cells into a patient’s brain to decrease cognitive impairments is not a practical treatment.

However, sprouting is a therapeutical target that could lead to a much greater increase in ACh. Although it is doubtful an increase in sprouting will lead to a complete recovery, a drug that could increase outgrowth of cholinergic neurons could greatly delay the progression of AD. There are many important questions to ask in this area, including to what extent the natural compensatory sprouting response is possible in old age. I examined this question with my thesis work.

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3. Compensatory Cholinergic Sprouting: An Analysis of Feasibility

3.1 The Question

In order to better understand my hypothesis and experiment, I would like to summarize the important points I have stated.

1. The cause of AD remains unknown, except in a small percentage of genetic

cases. Even in these cases, since it is not known what causes that genetic

mutation, there is no way to prevent the disease.

2. Existing therapies address symptoms and revolve around maintaining the

concentration of ACh for a longer time in the cortex by pharmacological

intervention.

3. The source of the cholinergic innervation to the cortex, the basal forebrain, is

severely damaged in AD, leading to cognitive decline.

4. Increasing ACh levels in the cortex by facilitating cholinergic neuron sprouting

of axon collaterals into denervated regions could compensate for the loss of

neurons.

With these important concepts in mind, my questions were: Can the BFCN remaining after damage sprout and re-innervate the EC? If the response is possible, is this ability to sprout maintained with increasing age? If we were able to document a compensatory response, even to a small extent, this finding would suggest a therapeutic window of opportunity at which point an intervention could be successful to maintain normal levels of cholinergic innervation.

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3.2 Hypothesis

My working hypothesis was that BFCN in the rat are able to mount a compensatory response in reaction to a small lesion. However, I also anticipated that age may reduce the extent of such a sprouting response. If this was the case, the adult

(12-20 months old) would show a reduced compensatory response in cortical cholinergic innervation following an immunotoxic lesion in comparison to young rats

(3 months old), a reduction that would be more pronounced in the aged (>20 months old) rats.

3.2 Overview and Rationale

We asked whether BFCN are able to exert compensatory sprouting and re- innervate an area of the cortex. To address this question we chose an experimental model in which rats 3 months old, 12-15 months old, 18-20 months old, and >24 months old would receive a small cholinergic specific lesion in the horizontal diagonal band of broca (HDB) on one side of the brain. Some of the HDB neuron cell bodies died, causing their axons to wither away, denervating areas of the EC (Fig. 4, 5). Other

HDB neurons survived. After survival periods of 2 weeks, 8 weeks, 12 weeks, and 24 weeks for different rats, cholinergic fiber density was measured in the EC to provide evidence for a sprouting response when compared to the contralateral, intact side of the brain.

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3.3 Rat Models and Aging Effects:

Animal research is an important step in obtaining information about human diseases and possible treatments. In order to gain a better understanding of sprouting, we obtained rats from the National Institute on Aging (NIA) for use in this study.

There are several rat models that can be used in this experiment, but the Fischer 344

(F344) rat is one of the most common models to use in aging studies, and has been supported by the NIA since its inception (Gallagher, Stocker, and Koh 2011). I used male F344 rats, 3 to 30 months old, to gather a lifetime study. Due to differences in rat strains, it is difficult to predict exactly how the age of rats compares to that of humans.

However, it is generally accepted that 3-12 months is considered young, 12-20 months is considered adult to middle aged, and 20-30 months is considered aged (Gallagher,

Stocker, and Koh 2011).

There have been controversies regarding dietary restrictions changing life span, and kidney lesions (Shimokawa, Higami, and Hubbard 1993) in old age, but other models, such as the Sprague-Dawley strain, have shown significant problems in old age (Fischer et al. 1992), including cholinergic neuron cell death. The death of cholinergic neurons has not been described in aging rats of the F344 strain, and, furthermore, other studies have reported a shrinkage in cell size, but not cell death, with age (Mesulam, Mufson, and Rogers 1987). It was important for me to use a rat strain that does not have significant cholinergic issues with normal aging. We created a model of some of the cholinergic damage done by AD by inducing a lesion. We did not want other normal aging processes to influence the results. Therefore, we picked Carnes 27

the F344 rat. To avoid a possible confounding factor with hormones, we used only male rats (Ábrahám et al. 2009). Taking these factors into consideration, we believe male F344 rats were the correct model choice.

3.4 Experimental Induction of Cell Loss

Other studies (Casamenti et al. 1988) have used non-specific lesioning tools, such as ibotenic acid, which kills both cholinergic and non-cholinergic cells. The study did not find a compensatory response in aged rats.

Others have examined sprouting using excitotoxins, which interfere with the function of another neurotransmitter, glutamate. This toxin acts by interacting with an

N-methyl D-aspartate (NMDA) receptor on the neuron receiving a signal. It allows excessive amounts of calcium ions to enter the cell, activating enzymes (Manev et al.

1989) that then go on to destroy the nerve cell. This process, however, has been shown to interact with the expression of genes that may help with the regenerative response (Leah, Herdegen, and Bravo 1991), and does not directly target cholinergic neurons. Based on earlier results from our group (Hartonian and de Lacalle 2004), we speculated that a small dose of 192-IgG Saporin immunotoxin will spare some cholinergic neurons in the area and drive compensatory sprouting.

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3.5 Description of 192-IgG Saporin

Fig. 6 – Immunotoxin mechanism

Example demonstrating the internalization of an immunotoxin. 192 IgG-Saporin includes an antibody targeted to cholinergic neurons. Saporin is a ribosome inactivating protein (RIP) which is eventually released and leads to protein synthesis inhibition and eventual cell death (Srivastava and Luqman 2015).

In our study, rats received a small injection into the HDB using stereotaxic coordinates of the rat brain. The injection was an immunotoxin, 192-IgG-Saporin, which caused a lesion. The compound is made up of a monoclonal antibody against the low-affinity nerve growth factor receptor, also called p75NTR, combined with a cytotoxin, saporin. In the basal forebrain, p75NTR is only expressed on cholinergic neurons. When the immunotoxin is injected into the brain, the antibody binds to p75NTR, is internalized, and then separates from saporin. Saporin is a ribosome Carnes 29

inactivating protein, having N-glycosidase activity. This disrupts the formation of proteins, leading to cell death (Book, Wiley, and Schweitzer 1994).

Several studies (Schliebs, RoBner, and Bigl 1996; Leanza et al. 1996) have also employed this technique and confirmed that 192-IgG Saporin can be used to selectively kill cholinergic neurons in the basal forebrain by one week after injection.

Injection of 192 IgG-Saporin into the basal forebrain therefore mimicked the loss of cholinergic cells, a component of AD.

3.6 Rationale for the Location of the Lesion

The lesion was administered in the right HDB. Axons from these neurons project into the EC (Fig. 3, 4). The HDB and the EC have only ipsilateral projections, meaning there were no projections from the lesioned right side that projected to the left EC (Fig. 4). This lack of contralateral innervation has been found in the basal forebrain in several tracer studies through the MS and the nucleus basalis (Bigl,

Woolf, and Butcher 1982; Price and Stern 1983) , and was confirmed for the HDB

(Dinopoulos, Parnavelas, and Eckenstein 1986) through a morphological characterization of neurons which found no contralateral innervation. This allowed us to use the spared left side as a control when measuring fiber density.

3.7 Rationale for the Choice of Histochemical Technique

To visualize fibers in the EC, I used AChE histochemistry following a protocol adapted from similar work that was published earlier (Hartonian and de Lacalle 2004). Carnes 30

As mentioned earlier, AChE is an enzyme that breaks down the neurotransmitter ACh, and is found around cholinergic neurons in large quantities. I used choline acetyltransferase (ChAT) and p75NTR immunohistochemistry to visualize cell bodies in the HDB, as both proteins can be commonly found in the cholinergic neuron cell body

(Hartonian and de Lacalle 2005). ChAT, which I have previously mentioned, is an enzyme synthesized in the cell body of the neuron involved in the synthesis of ACh.

Immunohistochemistry with antibodies against ChAT, and p75NTR, and AChE histochemistry are techniques that can be employed by neuroscientists to determine the presence of ChAT, p75NTR and AChE, respectively. In the basal forebrain, only cholinergic neurons express the p75 receptor, making it an excellent marker for cell bodies in the HDB (Hartonian and de Lacalle 2005). ChAT immunohistochemistry is also a useful method to identify cell bodies in the HDB, and we used this to confirm our p75NTR immunohistochemistry findings.

These two target proteins are not as useful the identification of the axon fibers of cholinergic neurons, however, as discovered in previous research (Hartonian and de

Lacalle 2005; Lysakowski et al. 1989). Therefore, we used AChE histochemistry to stain these fibers. AChE does not prove a neuron is a cholinergic neuron in the cortex, but AChE has been shown to correlate directly with cholinergic innervation (M.

Mesulam, Mufson, and Rogers 1987).

How does the staining work? In the AChE staining technique, an enzymatic reaction reduces ions which trap Cu2+, forming a precipitate. This leads to the visualization of neuron fibers and cell bodies. The precipitate, called Hatchett’s Carnes 31

Brown, can have enzyme activity similar to that of peroxidase, which will cause unspecific background staining. (Tago, Kimura, and Maeda 1986) To prevent this, a blocking step is included in the procedure to stop this activity, allowing for specific visualization. Successful staining will allow me to quantify the fiber density in the EC in both the lesioned and non-lesioned areas of the rat.

To stain ChAT and p75NTR proteins, an immunohistochemistry technique was used. Immunohistochemistry involves using antibodies that will bind to an antigen. A primary antibody binds to the protein, which is the antigen for the primary antibody.

Then secondary antibodies are used that are made to recognize the primary antibody as the antigen. The secondary antibodies, which are now bound to the primary antibody, are often conjugated to an enzyme such as horse radish peroxidase. This enzyme can be utilized in a chemical reaction to create a colored product which allows for the visualization of the target protein location. This method allowed us to quantify the number of cell bodies in both the lesioned and non-lesioned sides of the HDB.

3.8 Rationale for the Choice of Image Analysis Technique

To determine the fiber density of the stained EC tissues, we took pictures of three different EC slices from the same rat using a digital camera attached to a microscope. The same was done to determine the cell body count in the HDB. For the

EC fiber counts, the images were overlaid with a grid using Photoshop software (Fig.

7). We quantified fiber density in lesioned and intact sides using a 230 µm x 200 µm grid. We then counted the number of points at which the AChE-stained fibers intersect Carnes 32

Fig. 7 – Grid overlaid on stained EC tissue

A measurement of fiber density can be calculated from the number of times any stained fiber intersects the grid (blue arrows) overlaid on the stained EC tissue images (Hartonian and de Lacalle 2004). the grid lines and these numbers were used as an indirect measure of fiber density. In the HDB, we counted cell bodies in lesioned and intact sides as well. Since the HDB is continuous with the VDB and MS, it is important to count the cell bodies that form a noticeable band, which will be discussed in the methods.

There are numerous ways to go about counting stained fibers in the EC and cell bodies in the HDB. We followed the system mentioned above, which was developed previously in my lab and set up due to the requirements of the project.

Stereology is a commonly used counting method that has gained traction in neuroscience over the past decades. However, we decided, in agreement with others

(Guillery 2002; Hyman and Gomez-Isla 1998), that stereology, depending on the level of accuracy required, is not necessary. Stereology has two main tenants, which are that Carnes 33

volume must be used instead of density to prevent counting biases due to atrophy, and that certain methods must be utilized to prevent double counting and bias. The method used in my lab addresses the major goals of stereology while using a simple procedure.

Using the contralateral side of the brain as a control makes all cell and fiber counts relative, removing any density measurement issues that would be caused by atrophy. The tissue sections were cut 40 microns thick, and the average size of cell bodies in the HDB are about 20 microns in diameter. Due to the much smaller size of cells compared to slice thickness, double counting was not a significant issue. The sample size was also increased by taking 3 sections of the HDB and EC on both the spared and lesioned side, at least 480 microns apart from each other. This increased the sample size and helped prevent bias and double counting. The grid was overlaid in the lateral EC, where the HDB neurons are known to project, and the cell counts in the

HDB are completed at the location where the lesion was administered. Stereology requires sections in unequal places at random. Given the heterogeneity of the basal forebrain, taking random, unequal sections would lead to incorrect assumptions. Our method also used relative numbers to the contralateral side of the brain, instead of absolute numbers acquired in stereology. The method we used was much less complicated and intensive than following strict stereological methods, but still allowed us to achieve the level of accuracy required by addressing major points of stereology.

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3.9 Data Analysis Tools

To finish my project, I used statistical analyses, which allowed me to determine whether the data gathered is representative of a population, or if the results are due to chance. I anticipated applying t-tests to compare changes in fiber density in the lesioned side versus the spared side, and ANOVA to compare fiber density across time and across age groups. This was a very important part of the project for me, as I lacked hands-on experience with statistical methods. Statistics is a key area of all scientific research, and a deeper understanding will be important in any future studies

I conduct as a physician.

4. Significance:

This research is important to the field of AD. The progression of AD correlates with a decrease in cortical cholinergic innervation, particularly in the basal forebrain, cerebral cortex, and hippocampus (Hartonian and de Lacalle 2004). This makes the

EC cholinergic fibers and the neuronal cell bodies located in the HDB key elements in

AD. In 1991, it was suggested using morphological studies and staining techniques, that the EC was an important point in the progression of AD (Braak and Braak 1991).

In fact, a recent study (Khan et al. 2014) using MRI imaging shows that amyloid deposition and neurofibrillary tangles most likely start in the EC in AD.

As mentioned earlier, many research groups are focused on elucidating the causes of amyloid deposition and of neurofibrillary tangles, but we are still far from Carnes 35

conclusive answers. With regards to the mechanisms that cause cholinergic neurodegeneration, they remain unclear, although there are a number of potential explanations. My work is significant because it does not focus on the mechanism of disease, but on finding whether there is a way to compensate for this degeneration.

It is important to discover whether compensatory changes will occur in the

HDB and EC in aged rats, as the HDB and EC are important in memory and AD. If there are temporary or long term compensatory changes in the HDB and EC of these rats, the results would suggest that there is a potential for therapeutic methods that could preserve this compensatory response. If there is no compensatory response, it will still increase our understanding of the EC. Either way, the completion of this research will help fill a gap of knowledge about an area of the brain that is of immense importance to AD.

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METHODS

1.1 Subjects:

Male F344 rats were obtained from the National Institute of Aging colony at

Harlan in Indianapolis, Indiana. In the 3 mo group, 29 rats were used, in the 15-20 mo group, 26, in the 18-20 mo group, 27, and in the ≥24 mo group, 22 rats were used. The rats were kept in cages, two per cage, and followed a 12 hour light/dark cycle. They were given food and water ad libitum. Work with the rats was completed at Harvard

University and California State University Los Angeles, following approved institutional animal care protocols were followed. (Hartonian and de Lacalle 2005)

1.2 Administration of the Immunotoxic Lesion:

The lesion was given after the administration of anesthesia with intraperitoneal injection of 0.3ml/100 g of Equithesin. The rats were placed in a Kopf stereotaxic frame. This frame is used in stereotaxic surgery, which is a minimally invasive surgery that requires 3D coordinates to specifically target small areas, and in the case, the right

HDB. The skin was opened and holes were made in the skull using a small drill.

Infusion of 140 nl of .075mg/ml of 192 IgG-Saporin was then conducted using a glass micropipette with an air pressure injection system under aseptic conditions. In order to maintain the correct angle for injection, the incisor bar was set to 3.3 mm below the interaural line, or the line between the ears. The three coordinates in relation to the bregma were as follows: anterior-posterior, -0.4 mm, medial-lateral, +2.0 mm, and dorsal-ventral, -8.7 mm (Paxinos and Watson 1998). Carnes 37

1.3 Tissue Collection:

Within each age group, rats were sacrificed at either 2, 4, 8, 12 or 24 weeks post-lesion. Under profound anesthesia with 7% chloral hydrate, rats were perfused with 100-150 ml of saline, 250 ml of 4% paraformaldehyde in phosphate buffered saline (PBS) with a pH of 6.5, 250 ml of 4% paraformaldehyde in PBS with a pH of

8.0, and 200 ml of 20% sucrose. The brains were dissected and then cut 40 microns thick with a freezing stage microtome. The tissue sections were collected into wells with saline in 1:12 series.

1.4 Staining:

Histological techniques were used to stain the tissue for ChAT and p75NTR to visualize cholinergic cell bodies. An AChE histochemistry protocol was used to stain for AChE to visualize cholinergic axon fibers.

For ChAT (Fig. 8) and p75NTR (Fig. 9) immunohistochemistry, a horse radish peroxidase-3,3’diaminobenzidine (HRP-DAB) protocol was followed. First, the tissue was placed in a solution of PBS, 0.25% Triton X-100, and 3% H2O2 for 30 minutes to prevent endogenous peroxidase activity. Then the tissue was washed using a solution of PBS, 5% non-fat dry milk, and Triton X for one hour. The tissue was then placed in a dilution medium with the primary antibody (ChAT, UO95, 1:20,000 and for p75NTR, clone 192, 1:7,500), and kept at 4 °C overnight. Carnes 38

A.)

B.)

Figure 8 – Examples of ChAT immunohistochemically stained cholinergic HDB cell bodies in SPR 112. Pictures were taken using the 4x objective. A.) represents the control side, while B.) represents the lesion side of the HDB. The scale at the bottom left hand corner is 25 microns.

The tissue was then washed with PBS before being placed in a biotinylated secondary antibody for one hour. After this, the tissue was washed with PBS and then placed in an ABC complex (Elite Kit, Vector Labs) in PBS, diluted to a concentration of 1:1000 for one hour. The tissue was washed with PBS again before starting the

DAB step. Here, the tissue was added to a solution with buffer, 0.05% DAB, and

0.01% H2O2 for eight to twelve minutes, followed by three washes with buffer Carnes 39

A.)

B.)

Figure 9 – Examples of p75NTR immunohistochemically stained cholinergic HDB cell bodies in SPR 312. Pictures were taken using the 4x objective. A.) represents the control side, while B.) represents the lesion side of the HDB. The scale at the bottom left hand corner is 25 microns. solution. After the last wash, the tissue was mounted on a glass slide and cover slipped. Controls were also conducted in parallel, following the same protocol except that the primary or secondary antibody was omitted. This was done to determine the specificity of the staining.

An AChE histochemistry with silver intensification technique was used to visualize cholinergic axon fibers (Fig. 10). First, the tissue was rinsed two times, one Carnes 40

minute each in a 0.1M acetate solution, pH 6.0. Then the tissue was incubated in

Hedreen Green solution with acetylthiocholine iodide as the substrate for 45 minutes.

This was followed by rinsing the tissue five times for one minute each in 0.1 M sodium acetate, and then placed for one minute in a developing solution of ammonium sulfide. The solutions were then rinsed five times for one minute each in 0.1 M sodium nitrate, and then immersed for one minute in 0.1% silver nitrate. The tissue were

A.)

B.)

Figure 10 – Examples of AChE histochemically stained cholinergic axon fibers in the EC of SPR 294. Pictures were taken using the 4x objective. A.) represents the control side, while B.) represents the lesion side of the EC. The scale at the bottom right hand corner is 40 microns. Carnes 41

rinsed with 0.1 M sodium nitrate five times for one minute each and then 0.1M sodium acetate five times for one minute each. The tissues were finally mounted on a glass slide, allowed to dry overnight, and coverslipped.

The histological work up to this point was performed by a graduate student before I joined the lab. I enrolled in the cellular and microbiology laboratory techniques class to practice and gain familiarity with immunohistochemistry and other staining procedures to help me better understand this section of the project.

1.5 Data Analysis:

Twelve representative pictures of the stained tissue sections were taken for each rat. Six were of cell bodies in the HDB, and six were of axon fibers in the EC.

Within these groups, three pictures were taken of the control/spared side of the brain and three on the lesioned side. Pictures of the HDB and EC were taken using stereotaxic coordinates to locate each area. The HDB pictures were taken at three levels (Fig. 11) between anterior-posterior 0.7 mm and 0.20 mm and the EC pictures were taken at three levels between -5.80 mm and -6.80mm (Paxinos and Watson

1998). The pictures were taken with a digital camera attached to a microscope using the 4x objective for the HDB cell bodies and the 20x objective for the EC axon fibers. Carnes 42

A.) B.)

C.)

Figure 11 – Example of the pictures taken at three different layers of the HDB on the control side in SPR 312. These are stained using p75NTR immunohistochemistry. Pictures were taken using the 4x objective. A.) represents the anterior layer, B.) represents the middle layer, and C.) represents the posterior layer. The scale at the bottom left hand corner is 25 microns.

In the HDB, cell counts were done by tallying the total number of p75NTR cell bodies within the HDB in each side. The counts on both sides were averaged and a point value was calculated for the lesioned side as a percentage of the control

(Lesion*100/Control). To verify the effect of the toxin and obtain a measurement of the extent of the lesion that resulted, counts from the young group were compared to the adult/aged rats. Counts were obtained from the group of 3 mo rats (n=29). Cell counts from the >12 mo rats were pooled into a single group (n=8), including n=4 for

12-15 mo rats, n=2 for 18-20 mo rats, and n=2 for ≥24 mo rats. Carnes 43

In the EC, an indirect measure of fiber density was calculated. The EC images were overlaid with a 230 µm x 200 µm grid using Photoshop software (Fig. 12). The points where fibers intersect the grid were counted and the total number of

A.)

Fig. 12– Counting grid overlaid on stained EC tissue

In A.) the grid (highlighted in red) is placed on the image in a similar location to the contralateral side image. B.) represents the grid with fibers that intersect the grid marked by a yellow dot. The scale in the bottom left hand corner is 40 microns. The pictures were taken with the 20x objective. Carnes 44

intersections were used as an indirect measure of fiber density. The total number of intercepts counted on all 3 slides of each side were averaged and a point value for each subject was calculated and the lesioned side was represented as a percent of the control.

1.6 Statistics:

Paired T-tests were used to compare the cell counts between the control and lesioned side of the young (3 mo) and aged (12-27 mo) rats. A paired T-test was also used to compare the difference in the effect of the lesion on the HDB cell count between young and aged rats by using a percent of the control side. Several paired T- tests were applied to examine whether the lesion had an effect on the axon fiber density in the EC by comparing the control and lesioned sides in each survival time for each age group. An index was created by dividing the percentage of the control fiber density for each rat by the percentage of the control cell body count for the rat. This accounted for differences in the size or effect of the lesion. Then, using these numbers, one way ANOVA tests were conducted to compare the fiber density results across the post lesion survival times within age groups to determine whether sprouting occurred.

Carnes 45

RESULTS

1.1 Effect of the Toxin in the HDB:

Immunohistochemistry for p75NTR was used to determine the effect of the immunotoxin 192-IgG Saporin on cell bodies of the HDB (Fig. 13). These counts indicate that in both young and aged rats, the immunotoxin injection was successful in producing a lesion adequate to our goals, that is, a partial and well localized lesion

Y o u n g (3 m o )

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Fig. 13 – Cell counts of HDB cell bodies in the control and lesioned sides of the brain, 2 weeks after the lesion. A two-tail paired t-test was used and found a significant difference between both sides in the two groups (p < 0.05). The average of the counts in 3 sections of the HDB were employed. n=9 for the young group; n=8 for the aged group. Carnes 46

within the HDB to allow remaining neurons to sprout. Sample images of the lesion are shown in figures 8 and 9.

In the young group, 33% of the cholinergic neurons remained after the lesion.

In the aged group, the average was 59%. While there is substantial cell loss in both groups, it is far more pronounced in the young rats.

1.2 Effect of the lesion in the HDB on the EC:

We used an enzyme histochemistry technique to label AChE fibers in the EC, so that we could indirectly measure the effect that a lesion at the cell body would have on the axons. First we quantified fiber density as described in the methods section, and compared the results from the lesioned side versus the control, spared side. As expected, all 19 groups (including all survival time groups in each age group) had statistically significant differences in fiber density between the lesion and control sides

(Fig. 14-17). Two-tailed paired t tests were used to make these comparisons, and in all cases p < 0.05. This analysis indicates that loss of cholinergic neurons in the HDB led to a decrease in cholinergic axon fiber density in the EC.

Carnes 47

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Fig. 14 – Effect of the immunotoxin 192-IgG Saporin on cholinergic axon fibers in the EC of 3 month old rats. Fiber density in the side ipsilateral to the lesion was compared to the contralateral, control side of the EC. Two tailed paired t-tests were used. As in the 3 mo post lesion survival groups shown here, all other age and post lesion survival groups had a significant difference in density. * signifies p < 0.05

Carnes 48

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Fig. 15 – Effect of the immunotoxin 192-IgG Saporin on cholinergic axon fibers in the EC of 12-15 month old rats. Fiber density in the side ipsilateral to the lesion was compared to the contralateral, control side of the EC. Two tailed paired t-tests were used. * signifies p < 0.05 Carnes 49

t 5 0 0 n t 8 0 0 *

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Fig. 16 – Effect of the immunotoxin 192-IgG Saporin on cholinergic axon fibers in the EC of 18-20 month old rats. Fiber density in the side ipsilateral to the lesion was compared to the contralateral, control side of the EC. Two tailed paired t-tests were used. * signifies p < 0.05 Carnes 50

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Fig. 17 – Effect of the immunotoxin 192-IgG Saporin on cholinergic axon fibers in the EC of ≥ 24 month old rats. Fiber density in the side ipsilateral to the lesion was compared to the contralateral, control side of the EC. Two tailed paired t-tests were used. * signifies p < 0.05 Carnes 51

The effect of the lesion was maintained up to 24 weeks (the longest time point examined) as shown in Figure 18. At 12-15 months of age, fiber density does not increase beyond initial lesion levels, but there is a fiber density increase between 4 and

12 weeks post-lesion survival.

At 18 months of age and beyond, there was no recovery in fiber density (Fig.

18, C-D), in contrast with findings in the young rats (Fig. 18, A).

A.) 3 M o n th O ld R a ts B.) 1 2 -1 5 M o n th O ld R a ts

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0 F F 2 4 8 1 2 2 4 2 4 8  1 2 P o s t le s io n s u rv iv a l tim e (w e e k s ) P o s t le s io n s u rv iv a l tim e (w e e k s )

Fig. 18 – Changes in fiber density in the EC, ipsilateral to the lesion in the HDB, expressed as a percent of the spared, contralateral side. (100*Lesioned Side Fiber Density/Control Side Fiber Density). The mean fiber density for each survival time is depicted. In each group, n ≥ 4. One way ANOVA’s were conducted to compare fiber density across post-lesion survival times. No statistically significant differences were found. Carnes 52

Even after allowing for 24 weeks, fiber density in the middle aged (18-20 mo) and aged (≥24 mo) rats remained at or below the levels measured 2 weeks post-lesion.

This suggests that after adulthood (~12 mo), the sprouting capacity of the cholinergic fibers in the EC is no longer present.

Carnes 53

DISCUSSION

Our results demonstrate that aging abolishes the cholinergic sprouting response described in the young rat (Hartonian and de Lacalle 2005). While in 3 mo rats, there was an increase in fiber density from 2 weeks to 24 weeks post lesion of 10.8%, none of the other groups between 12 and 24 months old showed any recovery in fiber density between 2 and 24 weeks post lesion. However, in the 12-15 mo group, the more extensive denervation occurred 4 weeks after the lesion, and there was an increase in fiber density of 21.5% between 4 weeks and 12 weeks post lesion. In 18-20 and ≥ 24 mo rats, there was a further decrease in fiber density, which may be related in a similar way to the 12-15 mo group.

The delayed maximum denervation was an interesting finding that implies the toxin did not kill the cell bodies within 2 weeks, leading to further decrease in fiber density. This may be similar to the “penumbra effect” in stroke, where surrounding cells are damaged and die after the initial injury occurred (Fisher 2004) due to damage and loss of connections that may have been supported by cholinergic neurons that died. It could also have to do with the half-life of the immunotoxin. The half-life of

IgG antibodies on average is around 25.8 days (Mankarious et al. 1988) and this would most likely explain how the lesion was not complete in two weeks. This may be an interesting area for further research on the toxin.

Another interesting finding was the effect of the toxin in the HDB when comparing young and aged rats. On average, 26% more cholinergic cell bodies survived in aged rats. Further review of the literature revealed that p75NTR, the target Carnes 54

for our immunotoxin, has decreased expression with increasing age in the rat (Cowen et al. 2003), which corresponds with decreased neurotrophic factors (Silhol et al.

2005). This does not affect our results however, as we used the contralateral side of the brain within a rat as a control in all cases, which negates the influence of differences within the rat, such as a decrease in receptor expression, or age related death of cholinergic neurons.

Very early studies (Casamenti et al. 1988) examined recovery responses in the basal forebrain of aged rats following the use of large, non-cholinergic specific lesions that caused large amounts of complete neuronal death. The authors found no recovery in cholinergic activity with bilateral lesions, and only partial recovery with unilateral lesions. It is not clear how death of cholinergic and noncholinergic neurons affects a potential sprouting response.

More recently, other studies have shown that sprouting can occur in the brain of young animals (Hartonian and de Lacalle 2005; van Groen, Miettinen, and Kadish

2011). However, another recent study (Szigeti et al. 2013) found no long term compensatory changes in cholinergic innervation in the primary motor cortex and somatosensory cortex of the rat 20 weeks post-lesion. The main difference of this work compared to ours is that in this work, they used a very large amount of 192-IgG

Saporin (0.5 μl compared to our 140 nl) which induced an almost complete lesion of the target area, thus leaving very few neurons (~10%) to exert a compensatory sprouting response. We think that with this small number of neurons left, there may be too few neurons and too much sprouting required by each neuron for the observable Carnes 55

occurrence of sprouting. This work also differs from ours in the location of the lesion, and the cortical area examined. Furthermore, this study used young adult rats of a different strain. Compensatory sprouting was limited in the aged brain in our study but we postulate that there may be a “window of opportunity”, or a time when compensatory sprouting is still present, in 12-15 mo adult rats. This could be an important target in preclinical AD, which will be discussed later.

Neither ours nor the other studies have investigated whether sprouting has a functional effect. There have been many studies looking at the recovery of enzymatic activity involved with cholinergic neurons, which could imply a recovery in function.

These studies looked at AChE and ChAT levels. They demonstrated mixed results, with some finding full recovery (Belleroche, Gardiner, and Hamilton 1985; Gardiner et al. 1987; Cossette et al. 1993), others finding partial recovery (Wenk and Olson

1984) , and others, no recovery (Henderson 1990; Ojima, Sakurai, and Yamasaki

1988; Casamenti et al. 1988; Bartus et al. 1985). These studies examined different areas of the basal forebrain and had varying methods, including non-specific lesioning tools which could cause widespread cell death. This may have led to the mix of results for whether there is a recovery of function. Taking this in to account, these works have led us to believe that recovery of function after sprouting is possible, and future work will hopefully elucidate this further.

To better understand the impact of aging on the potential for compensatory sprouting, as well as to explore if there truly is a window of opportunity for this mechanism at the 12-15 mo stage, we need to correlate the extent of cell body loss Carnes 56

with the extent of fiber density within each animal in the study. Preliminary analysis

(non-quantitative) suggests that the lesion size was equally small in all subjects, and since fiber density did not increase with time/age, there is no reason to believe the lesions were too small. However, if cell loss in the adult animals were to be found too extensive, then an index of fiber density divided by cell counts may be helpful. Further work will be done to create this index and examine if this index paints a different picture than our results.

Carnes 57

FUTURE DIRECTIONS

There are inhibitory factors that prevent the repair of neurons in the brain

(Schwab 1996), including different signaling molecules. There are also molecules in myelin which impede sprouting (Schwab 1996a) . However, there are also targets such as nerve growth factor, which have been shown to facilitate sprouting in some cases

(Yasui et al. 2012). More research must be done in this field to determine what can be done and whether any of these molecules can be reasonably inhibited or promoted to facilitate sprouting in a clinically beneficial way. For example, it would be interesting to measure levels of nerve growth factor post-lesion to see if these levels are increased to encourage sprouting, and whether these levels decrease with age. It would also be interesting to use cell cultures and examine different molecules and whether they induce or inhibit sprouting in vitro. If the 12-15 mo rats maintained the ability to sprout as suggested by our results, then research in this area could be of critical importance to finding a treatment for cholinergic cell death in AD.

Another important area of research in relation to this work is the identification and proper diagnosis of preclinical AD. There have already been studies examining the use of MRI and morphological methods to help diagnose preclinical AD, as well as the use of biomarkers (Cowen et al. 2003). Finding the best way to use these findings to diagnose patients in a clinical setting would go a long way in getting early care for patients. As suggested by our results in young and adult rats, a sprouting response may be possible. Amyloid deposition can begin 20 years prior to any symptoms of AD

(Mayo Clinic, 2016). Although the age ranges of the F344 rat are rough estimates, Carnes 58

identifying preclinical AD as an adult, possibly around 40-50 years old, could lead to treatments that facilitate sprouting. Our hope is that one day, a patient can be identified and treated before AD progresses, adding many happy, memory filled years to their life.

Carnes 59

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