國立臺灣師範大學生命科學系碩士論文

氣體傳遞物質硫化氫對於阿茲海默氏症

的神經保護功效

Neuroprotective effect of H2S gasotransmitter on

Alzheimer's disease mouse model

研 究 生：陳 淑 玲

Shu-Ling Chen

指導教授：謝 秀 梅 博士

Hsiu-Mei Hsieh

中 華 民 國 103 年 7 月

致謝

這篇論文涵蓋了我的研究所生活，從開始到結束。在這兩年的

研究日子裡，有許許多多的人不斷的支持、鼓勵著我，倘若沒有他

們的幫助，這篇論文無法如此順利的完成。

首先，感謝指導教授 謝秀梅老師兩年來的照顧及指導，老師悉

心的教導讓我進入神經科學領域並學習新知，藉著不時的與我討

論、釐清觀念並指引我正確的方向，且您認真嚴謹的態度著實令我

受益良多；在實驗之外，也感謝老師在這兩年日子裡的關心及照

顧，讓身處異鄉的我感受到家人般的溫暖。另外，感謝梁庚辰老師

及王慈蔚老師在口試時給予許多實驗以及思考邏輯上的建議，得以

讓這篇論文趨於完整。

接著，感謝 黃慧貞老師帶領我進入阿茲海默氏症的研究領域並

在實驗上提供許多的寶貴想法，引領我思考並嘗試著解決問題；感

謝薇琳學姐的傾囊相授，使我在實驗及文獻閱讀都能快速進入狀

況。私底下，妳們耐心的傾聽並與我分享生活大小事，使得我有更

多智慧去面對未來人生。

因為大家的指導，兩年間，我學會了許多事情，謝謝執中學

長、紫綾學姐在即將離開的那個暑假，仍不厭其煩的盯著我、提醒

著我學習與實驗技巧；小強，謝謝你總是在我撞牆的時候給我許多

實用的建議跟方法；鮪魚，謝謝你帶我進入細胞這個可愛的小世

界，以及美味甜點的分享；育晨，謝謝你貼心的下午茶，且偶爾的

一句加油總是讓人感到窩心；仁華學姐，謝謝你在細胞實驗上的大

力幫助以及人生經驗的分享。

還有我的實驗室夥伴：定中和翔文，謝謝在這兩年的日子中，

你們陪我一起卡關、一起面對再一起解決問題，也謝謝你們總是為

實驗室帶來笑聲，讓實驗生活不無聊；馬偕的學妹：朧芸、芷妍、

詩芸、于璇，謝謝你們犧牲假日時間幫忙照顧老鼠、隨時注意老鼠

的身體狀況，讓我在實驗上無後顧之憂；還有其他陪了我兩年的同

學與朋友們，謝謝你們願意聽我嘮叨實驗上的不順利，然後拍拍我

的肩，鼓勵我再努力往前邁進。

不可遺忘的要感激這篇論文的最大功臣：3×Tg-AD & B6 mice，

如果沒有你們的犧牲與奉獻，論文絕對沒有完成的一天，因此，我

要對你們致上最深的謝意。

最後，感謝我的家人，謝謝阿公、爸爸、媽媽、妹妹、弟弟們

體諒我的研究性質，包容我半年甚至一年才能回家。有你們的支持

跟鼓勵，我才能無所顧忌的往前，得以順利完成我的碩士學位，謝

謝你們。

Table of contents

中文摘要 ..................................................................................................... 1

Abstract ....................................................................................................... 3

Introduction ................................................................................................. 5

Materials & methods ................................................................................. 15

Results ....................................................................................................... 26

Discussion ................................................................................................. 40

References ................................................................................................. 45

Tables & Figures ....................................................................................... 54

Appendixes ............................................................................................... 99

1

中文摘要

目前阿茲海默氏症發病率持續上升，又缺乏有效治療方法的現

況已造成全球醫療嚴重的挑戰。近期有研究指出硫化氫對許多神經

退化性疾病如阿茲海默氏症具有抗氧化、抗細胞凋亡，以及抗發炎

的效果。然而，硫化氫、類澱粉蛋白，以及 tau 蛋白之間的關聯性

尚未闡明。在此研究中，我們將以 oligomeric Aβ42 處理的海馬迴初

級神經元培養系統與給予壓力的阿茲海默氏症基因轉殖鼠探討硫化

氫對於阿茲海默氏症的作用效果與機轉。首先，在建立海馬迴初級

神經元培養系統中確認培養到第 9 天時，海馬迴神經元之樹突開始

延展及分支;另外也發現 2 μM AraC 能抑制星型膠細胞之生長且對神

經元造成之傷害性較小。在確認了海馬迴初級神經元培養系統之條

件後，我們接著進行 1 μM oligomeric Aβ42 對細胞處理 0，0.5，1，

6 及 12 小時後之影響，發現 1 μM oligomeric Aβ42 培養 1 小時後會

造成細胞存活率下降、LDH 釋放量增加、成熟神經元數目減少、神

經突數目與長度減少及突觸密度下降等傷害，所以以 1μM

oligomeric Aβ42 培養 1 小時為一進行藥物評估之適合條件。另外，

不同劑量的硫化氫鈉（硫化氫供應物）與 oligomeric Aβ42 以前處

理、共同處理及後處理三種方式加入海馬迴初級神經元培養系統

內，我們發現 50 μM 硫化氫鈉前處理或共同處理 oligomeric Aβ42 及

2

10 μM 硫化氫鈉後處理可以保護海馬迴初級神經元培養的神經細胞

型態及突觸密度。在動物實驗上發現電刺激所產生的壓力對於 6 個

月大的阿茲海默氏症基因轉殖鼠僅造成有限之傷害。因此，此種電

刺激之強度及給予方式對於 6 個月大的阿茲海默氏症基因轉殖鼠可

能不足以產生嚴重之壓力。另外，硫化氫鈉之給予可以避免認知功

能之退化可能是經由 PERK/eIF2α 途徑所產生的抗氧化與抗發炎反

應於電刺激的阿茲海默氏症基因轉殖鼠。。因此，硫化氫對於阿茲

海默氏症或許是一個具有潛力的治療策略之選擇。

關鍵字：硫化氫，海馬迴初級神經元培養，阿茲海默氏症基因轉殖

鼠，電刺激，寡聚體 Aβ42

3

Abstract

The worldwide incidence of Alzheimer's disease (AD) is increasing

and creating an unsustainable healthcare challenge due to a lack of

effective treatment options. Recent evidence shows that H2S could exert

antioxidant, anti-apoptotic, and anti-inflammatory effects against

neurodegenerative disease such as AD. However, the correlation among

H2S, Aβ and tau protein in AD are not been fully elucidated. In this study,

the effects and molecular mechanisms of H2S in AD were investigated in

hippocampal primary neuronal culture and APP/PS1/Tau triple transgenic

(3×Tg-AD) mice with inescapable foot-shock stress. At first, we

established the hippocampal primary neuronal culture as an in vitro

platform for a primary study. The neuronal processes were extended and

branched on 9 days in vitro (DIV). In addition, the medium with 2 μM

cytosine arabinoside (AraC) inhibited the population of astrocyte

associated with limited neuronal damage. We then evaluated the effects of

1 μM oligomeric Aβ42 on hippocampal primary neurons with cultivating

timing from 0.5 to 12 hr. We found that 1 hr treatment induced total cell

loss and lactate dehydrogenase (LDH) released, and mature neuronal

number, neuritic length, processes, branches, and synaptic density

decreased. Therefore, treatment of oligomeric Aβ42 for 1 hr is an

optimized timing for evaluation of drug treatment. Different

concentrations of NaHS (an H2S donor) were applied to oligomeric Aβ42-

treated hippocampal primary neuronal culture in pre-, co-, or post-

treatment manners. We further found that NaHS pre-/co-treatment (50

μM) and post-treatment (10 μM) could effectively attenuate oligomeric

4

Aβ42-induced toxicity. We then evaluate the in vivo effect of NaHS on

the 3×Tg-AD mice associated with inescapable foot-shock stress. The

3×Tg-AD mice (6 month-old) with stress were administrated with either

NaHS or vehicle, and subjected to a series of behavioral evaluation (light-

dark transition test, open field test, elevated plus maze, and Morris water

maze). We found that inescapable foot-shock stress only induced limited

impact in 3×Tg-AD mice. However, the administration of NaHS

prevented the cognitive decline associated with anti-inflammation and

anti-oxidation via PERK/eIF2α pathway in 3×Tg-AD mice with mild

stress. Therefore, H2S is a potential therapeutic strategy to prevent the

cognitive dysfunction for AD.

Keywords: hydrogen sulfide, hippocampal primary neuronal culture,

3×Tg-AD mice, electrical foot-shock, oligomeric Aβ42

5

Introduction

Alzheimer's disease (AD) has become the most costly disease to the

society and a major public health problem in the world. The two

hallmarks of pathological characterization in AD are intracellular

neurofibrillary tangles (NFTs) composed of the tau protein and

extracellular deposition of plaques composed of β-amyloid (Aβ). There is

no disease-modifying treatment for AD, with current approved drugs

targeting the neurotransmitter systems such as cholinergic (Winblad et al.,

2006, Winblad, 2009), and glutamatergic (Atri et al., 2008, Mecocci et

al., 2009) systems which improve symptoms but whose role in functional

recovery is still debated so far (Mangialasche et al., 2010). These

therapeutic limitations are: 1) numerous hypotheses for its etiology, but

none have been conclusive to date, 2) mutlifactorial pathogenesis, and 3)

the blood brain barrier (BBB) selectively restricts the blood-to-brain

paracellular diffusion of compounds. Therefore, many efforts to find the

effective therapeutic strategies under less side effects and cost will be

urgent for AD in now.

The role of Aβ in AD

The amyloidal cascade hypothesis, which suggests that the

deposition of the Aβ peptide, especially Aβ42 in the brain is a central

event in AD pathology, has dominated research for the past twenty years

(Karran et al., 2011). Recently, several attempts have been made to

reevaluate the amyloidal hypothesis and to suggest new directions in AD

research (Castellani and Smith, 2011, Goate and Hardy, 2012). Evidence

6

suggests that the toxic Aβ species might be represented by oligomers

rather than monomers, fibrils, or plaques (Aisen, 2005, Li et al., 2011)

and much research has also been devoted to the search for

pharmacological approaches to prevent Aβ oligomerization as a therapy

in AD (Aisen, 2005). Therefore, drug to destabilize oligomer

conformation of Aβ will be potential effective therapeutic strategy for AD

rather than only to attenuate the level of Aβ.

The role of tau hyperphosphorylation in AD

The microtubule associated protein tau highly expressed in the axons

of neurons is to stabilize and facilitate the assembly of microtubules.

Recent evidence shows that the therapeutic target on tau protein might

have more powerful than Aβ in clinical expression (Costanza et al.,

2012). Evidence also suggests that misfolded hyperphosphorylated tau

proteins played an important role in the synaptic dysfunction (Tai et al.,

2012). In addition, evidence further shows that Glycogen synthase

kinase-3 (GSK3), a tau kinase, might be involved in pathological tau

phosphorylation in AD brain (Spittaels et al., 2000, Lucas et al., 2001)

and Aβ deposition (Phiel et al., 2003). However, the interrelationship

among Aβ, tau hyperphosphorylation, and GSK3 kinase activity is still

controversy so far.

Glycogen synthase kinase-3 in AD

GSK3, a serine/threonine kinase, is known to regulate critical

cellular functions such as structure, gene expression, mobility, and

7

apoptosis. Evidence shows that GSK3 was implicated in the formation of

both senile plaques and neurofibrillary tangles (Jope and Johnson, 2004,

Giese, 2009). GSK3 is not only intimately involved in many aspects of

amyloid precursor protein (APP) metabolism and Aβ production, but also

contributes to Aβ-induced neuronal toxicity (Aplin et al., 1997,

Takashima et al., 1998, Kirschenbaum et al., 2001, Ryan and Pimplikar,

2005). In addition GSK3 can phosphorylate many sites on tau. In AD

brain, some of the same sites are abnormally hyperphosphorylated

(Hanger et al., 1998, Johnson and Hartigan, 1999). GSK3 may contribute

to the abnormal hyperphosphorylation of tau, and promote tau

aggregation and neurotoxicity (Spittaels et al., 2000, Fath et al., 2002,

Noble et al., 2005) . However, evidence also shows that the same

treatment in amyloid-depositing vs. tau-depositing mice presented the

opposite effects (Lee et al., 2012).

Inflammatory response and oxidative stress in AD

Inflammation in a systemic or central nervous system is a

pathological hallmark of AD and is characterized by activation of

microglia and astrocyte and altered production of inflammatory

mediators. Microglia activation seems to be an early event in AD amyloid

pathology. In fact, activated microglial cells associated with Aβ plaques

have been observed at early stages of plaque appearance in AD transgenic

models (Gasparini and Dityatev, 2008, Meyer-Luehmann et al., 2008). In

addition, the release of inflammatory mediators might precede

morphological changes of microglia and might be triggered by Aβ soluble

8

species before plaque formation (Ambree et al., 2006, Solito and Sastre,

2012).

Abundant pro-inflammatory cytokines, chemokines, complement

products, and oxygen radicals are present in AD brains (Wyss-Coray,

2006, Rojo et al., 2008). Several cytokines have been associated with AD

development and progression, such as interleukin 1 (IL-1), interleukin 6

(IL-6), transforming growth factor β (TGF-β) and tumor necrosis factor α

(TNF-α) (Griffin et al., 2000, Rodriguez Martin et al., 2000). In in vitro

study, stimulation with Aβ oligomers, but not with Aβ fibrils induces the

pro-inflammatory cytokine TNF-α released from adult microglia (Meyer-

Luehmann et al., 2008).

In addition, the Aβ possesses the ability to reduce Cu2+ and Fe3

+

towards Cu+ and Fe2+, respectively. The molecular oxygen can react with

reduced metals thus generating superoxide anion, which in turn combines

with two hydrogen atoms to form hydrogen peroxide (Hureau and Faller,

2009). Evidence further suggests that Aβ is synergistic with pro-

inflammatory cytokines to induce neuronal damage via reactive oxygen

species (ROS)-dependent pathways (Meda et al., 1995, Medeiros et al.,

2007). Furthermore, ROS scavengers such as catalase also reduce the

activation of nuclear factor kappa-B (NF-κB), a transcription factor

mediating immune and inflammatory responses (May and Ghosh, 1998).

Therefore, the inflammatory response and oxidative stress will be

evaluated in the study.

Endoplasmic reticulum (ER) stress in AD

9

ER is the principal organelle responsible for the proper

folding/processing of nascent proteins and perturbed ER function leads to

a state known as ER stress. Evidence shows that ER stress was involved

in the pathogenesis of AD (Barbero-Camps et al., 2014, Placido et al.,

2014). Unfolded proteins are recognized in ER via three classes of

sensors, inositol-requiring protein-1 (IRE1), protein kinase RNA-like ER

kinase (PERK), and activating transcription factor-6 (ATF6) (Schroder

and Kaufman, 2005). Evidence also shows the ER stress response could

be localized to dendrites, and be related to synaptic loss and axonal

degeneration (Murakami et al., 2007). In addition, the staining of p-PERK

and p-eIF2α were clearly increased in hippocampal neurons associated

with phosphorylated tau and GSK3β staining in AD (Hoozemans et al.,

2009). Another study further points out that p-eIF2α induced the β-site

amyloid precursor protein cleaving enzyme 1 (BACE 1) activity and

amyloid load in AD (O'Connor et al., 2008). Therefore, ER stress maybe

plays an important role in the pathogenesis of AD.

Hippocampal primary neuronal culture

Primary neural cultures allow continuous visual access for

morphological studies such as neuritic length, neuritic branching, and

viability. These cultures make individual living cells accessible to apply

chemical or pharmacological agents. Additionally, the relative

proportions of neurons and glial cells can be controlled, and different

patterns of neuronal connectivity are beginning to be studied with

developments in culture substrates. Therefore, primary neuronal culture is

10

an important research tool that can be applied on a cell-by-cell basis to

morphological and physiological evaluation in in vitro model.

The development of hippocampal neurons in culture had four stages:

1) hippocampal neurons extend a lamella after planting; 2) there were

several minor neuritis undergoing growth; 3) the minor neurites of

neurons grow continuously, becoming two to three times longer than the

other; and 4) the dendrites have begun to grow and branch (Appendix 1)

(Kaech and Banker, 2006). Therefore, the evaluation of treatment effect

was conducted at stage 4 in the study.

The 3×Tg mice (APP/PS1/Tau) with stress used as an animal model in

AD

The 3×Tg mouse (human APPswe × human PS1M146V × human

tauP301L; 3×Tg-AD) model of AD is unique in manifesting both amyloid

plaques and NFTs in the brain. Thus, this model recapitulates the

hallmark lesions of AD more closely than models that have only plaques

or tangles (Oddo et al., 2003).

The progressive increase in Aβ deposition is detected in

hippocampus and cerebral cortex of the 3×Tg-AD mice as early as 3-4

months of age. Synaptic transmission and long-term potentiation (LTP)

demonstrated to be impaired at 6 months of age. Aggregates of

hyperphosphorylated tau are detected in the hippocampus between 12-15

months (Oddo et al., 2004). Evidence further suggests that environmental

risk factors such as stress might accelerate the cognitive loss in 3×Tg-AD

mice (Devi et al., 2010, Maggio and Segal, 2011). Some reports showed

11

that stress affected not only the amygdala-dependent but also the

hippocampal-dependent behaviors (Huang et al., 2010) and led to a

further decrease in LTP (Grigoryan et al., 2014). Therefore, in this study,

stress with an inescapable foot-shock to accelerate the pathologic

progression of the 3×Tg-AD mice was performed in the study.

Therapy in AD

Clinical therapy in AD was included cholinesterase inhibitors

(CHEI), and NMDA receptor antagonist. There are some currently used

CHEI drugs: donezepril, rivastigmine, galantamine, tacrine, and

metrifonate. However, the clinical findings tend to have nausea, vomiting,

diarrhea, headache, decreased appetite, dizziness and many other side

effects (Hansen et al., 2008, Tan et al., 2014).

Impaired synaptic function has been linked with the AD pathological

process (Lacor et al., 2007). N-methyl-D-aspartate receptors (NMDA

receptors) are known to maintain the synaptic plasticity and contribute to

memory formation (Rezvani, 2006). The NMDA receptor is a tetramer

composed of two NR1 subunits and two NR2 subunits or less commonly,

two NR3 subunits. NMDA receptor activation leads to Ca2+ influx and

triggers downstream signal transduction. Calmodulin-dependent kinase

(CaMK) and cAMP response element-binding (CREB) protein are then

phosphorylated and trigger transcription of genes needed for LTP

formation. Evidence shows that LTP formation requires the activation of

NR2A, but not the NR2B subunit (Massey et al., 2004). In addition,

report indicated NR2B was required for long-term depression (LTD)

12

which played an important role of the old memory clearance. (Nicholls et

al., 2008, Foster et al., 2010, Malleret et al., 2010).Changes of

NR2A/NR2B ratio explained the effects on the kinetics of NMDA

receptor-mediated synaptic plasticity (LTD, LTP, and depotentiation)

(Hardingham and Bading, 2010). Furthermore, the NR2A/NR2B ratio has

been suggested to play an important role as a therapeutic index in the AD

(Cui et al., 2013). Therefore, the level of NR2A/NR2B ratio will be

evaluated in the therapeutic effect in AD.

Except CHEI and NMDA receptor antagonist, strategies attenuating

the amyloidogenic pathway by β-secretase inhibitor and γ-secretase

inhibitors have been under investigation for decades. However, the

development of β-secretase inhibitor turned out to be very challenging

due to problems of brain access, cell penetration, and oral bioavailability.

Therefore, the small molecules as β-secretase inhibitor did not show their

efficacy in cognitive improvement for AD patients in early clinical trials

and this study was also halted due to liver toxicity in phase III (Fan and

Chiu, 2014). In addition, recent evidence suggests that tau-center

treatment maybe play an important role of the therapy in AD

(Mangialasche et al., 2010) (Castellani and Perry, 2012). However,

neither Aβ- nor tau-center treatment can attenuate the impairment of

cognitive function in AD. Briefly, AD is not a one-gene, one-protein

disease and should be attributed to a network of interactions between

genes, proteins, organelles, cells, neurotransmitters, and the environment.

Those disease-modifying agents currently being developed typically

target one hypothesis and one protein. Thus, it is clear that a single drug

13

for the successful treatment of AD is not yet available. It is reasonable to

explore multi-target strategies and combination therapies.

The roles of gasotransmitter in organism

In the last decades, investigation of the pathophysiological and

pharmacological roles of “gasotransmitters” has represented a

challenging research field, which is still widely unexplored. An

endogenous gasotransmitter is characterized by the ability to diffuse

across biological membranes to modulate biological pathways/functions

at a physiological concentration, and the presence of specific biological

targets (Wang, 2002). These candidate gaseous neurotransmitters are NO,

CO, and H2S that shared properties, which qualify them as

gasotransmitters in that they 1) are small molecules of gas; 2) are freely

permeable across membranes and do not act via specific membrane

receptors; 3) are synthesized endogenously and enzymatically on demand

and their generation is regulated; 4) have well-defined specific functions

at physiologically relevant concentrations; and 5) their cellular effects

may or may not be mediated by second messengers, but these

gasotransmitters have specific cellular and molecular targets. Due to their

gaseous nature, these gasotransmitters are not stored in synaptic vesicle

and no presynaptic re-uptaken as other neurotransmitters. Therefore,

gasotransmitters are rapidly scavenged or enzymatically degraded after

their release to terminate their signaling activity, with biologic half-lives

on the order of seconds. An additional property shared by the three

gasotransmitters is their potential systemic toxicity at supra-physiologic

14

concentrations, which lead to the recognition of these gases as air

pollutants and toxins before their important in vivo functions were

identified. The most recent candidate to join the family of

gasotransmitters is H2S. H2S exhibits a number of distinct characteristics

compared with NO and CO. It has the greatest water solubility (Miller et

al., 2009, Kajimura et al., 2010). However, unlike CO and NO, H2S

makes an anionic conjugate base, with HS- as the predominant form at the

physiological of pH 7.4 (Kajimura et al., 2010). Furthermore, both CO

and NO can bind to the heme moiety, while H2S cannot. Therefore, H2S

might be a better candidate gasotransmitters than CO and NO. H2S is

involved in a multitude of physiologic functions, including immune and

inflammatory processes, perception and pain mediation (Kasparek et al.,

2008). H2S appears to confer cytoprotection via multiple mechanisms

including anti-oxidant and anti-inflammatory effects (Wang, 2003, Lefer,

2007). Furthermore, evidence also suggests that sodium hydrosulfide

ameliorated Aβ40-induced spatial learning and memory impairment,

apoptosis, and neuroinflammation in Wistar rats (Xuan et al., 2012).

Therefore, in the study, the effects and molecular mechanisms of the

exogenous H2S donor will be elucidated from hippocampal primary

neuronal culture to 3×Tg-AD with stress animal model.

15

Materials & Methods

Animals

The female pregnant C57BL/6J mice and 3×Tg-AD (harbouring

PS1M146V, APPSwe and tauP30IL transgenes) mice were purchased from the

National Breeding Centre for Laboratory Animals and the Jackson

Laboratory (004807), respectively. The 3×Tg-AD male mice (6 months

old) were randomly divided into four groups, with 12-15 animals in each

group: (i) non-stress with vehicle; (ii) non-stress with NaHS; (iii) stress

with vehicle; (iv) stress with NaHS. The mice were housed at 20-25℃

and 60% relative humidity under a 12-hr light/dark cycle, and food and

water were made available ad libitum. All experiments were performed

during the light phase between 7 AM and 7 PM. All experimental

procedures involving animals were performed according to the guidelines

established by the Institutional Animal Care and Use Committee of

National Taiwan Normal University, Taipei, Taiwan.

Experimental Timeline

For in vitro assay, embryonic day 16-18 (E16-18) embryos from

C57BL/6J female pregnant mice were sacrificed for primary hippocampal

neuronal cultures. To establish an in vitro artificial model of AD, 1 M of

A42 oligomer was applied to primary hippocampal neurons at DIV9.

After incubated with A42 for 0, 0.5, 1, 6, and 12 hr, respectively,

hippocampal neurons were collected for MTT assay and

immunocytochemical (ICC) staining of NeuN, Nestin, GFAP, MAP2 and

Synaptophysin (Fig. 1A). For in vitro assessment, different doses of

16

NaHS (1, 10, 50 or 100 M) (Sigma-Aldrich, St. Louis, MO, USA) was

applied to the primary hippocampal neuronal culture at DIV9 30 min

before (pre-treatment), after (post-treatment), or at the same time (co-

treatment) with Aβ42 oligomer. After culture for 1 hr with Aβ42 oligomer,

the hippocampal neurons were harvested to perform MTT assay and ICC

staining (Fig. 1B).

For in vivo assay, male 3×Tg-AD mice (6 months old) were

randomized to receive stress or sham treatment on days 6 and 7. In

addition, two groups of mice were administrated either vehicle or NaHS

(2.5 mg/ kg/ twice a day) via intraperitoneal injection during days 1-8, 13,

20, and 31. Subsequently, mice were subjected to a series of behavioural

evaluation in open field test, elevated plus maze (EPM), light-dark

transition test and Morris water maze (MWM). Mice were sacrificed for

enzyme-linked immunosorbent assay (ELISA), western blot and

immunohistochemistry (IHC) analyses after MWM (Fig. 1C).

Primary hippocampal neuronal culture

We used C57BL/6J mouse strain for primary neural culture. E16-18

embryos were sacrificed to isolate hippocampus under surgical

stereomicroscope. Tissues were trypsinized (0.05%) for 15 min in 37℃

and cells were cultured in neurobasal plating media [neurobasal media

(GIBCO, Carlsbad, CA, USA. ) containing 2% B27 supplement

(GIBCO), 0.5 mM glutamine (GIBCO), 25 μM glutamate (Sigma-

Aldrich), penicillin/streptomycin (GIBCO, 20 unit/ml), 1 mM HEPES

(Sigma-Aldrich), 1% heat inactivated donor horse serum (GIBCO)] in a

17

density (3×104 cells/cm2) onto poly-L-lysine (100 μg/ml) coated plates.

Half of the culture media was changed on DIV 1, 4 and 7, respectively

with fresh media without horse serum. To reduce glial cell population, 2

μM cytosine arabinoside (Sigma-Aldrich) was added on DIV 4 and 7.

Preparation of Soluble Aβ42 Oligomer

Oligomeric Aβ42 was prepared as previously described (Kayed et

al., 2003). White lyophilized Aβ42 powder (AnaSpec, San Jose, CA,

USA) was dissolved in hexafluoroisopropanol (Matrix Scientific,

Columbia, SC, USA), diluted with ddH2O and centrifuged for 15 min at

14,000 × g at room temperature. The supernatant (adjusted to pH 2.8-3.5)

was transferred to a new siliconized eppendorf and subjected to a gentle

stream of N2 for 10 min to evaporate the hexafluoroisopropanol. The

mixture was then stirred at 500 rpm using a Teflon coated micro-stir bar

for 48 hr at 22℃. The oligomeric Aβ42 solution was applied to the

culture with a final concentration of 1 μM under serum-free condition.

Vehicle was prepared in the same procedure only without Aβ42 powder.

The oligomerization of the Aβ42 has been previously confirmed by dot

blot and low molecular weight native gel electrophoresis.

Immunocytochemical (ICC) staining and high-content screening

Cells were harvested for ICC staining at different time-course after

oligomeric Aβ42 or/and NaHS treatment. The cells were first fixed with

ice-cold 4% paraformaldehyde (PFA, Sigma-Aldrich) for 30 min and

washed with phosphate buffered saline with triton X-100 (PBST) 3 times

18

for 10 min each. Nonspecific epitopes were blocked by 10% fetal bovine

serum (FBS) diluted by primary antibody diluting solution for 2 hr. Cells

were then incubated with primary antibody (Table 1) for 16 hr at 4℃. To

terminate the primary antibody reaction, cells were washed with PBST 3

times for 10 min each. Cells were incubated with fluorescence tagged

secondary antibody for 2 hr on 37℃. Cells were washed 3 times with

PBST for 10 min each to terminate secondary antibody reaction. Finally,

nuclei of cultured neurons were counter-stained with DAPI (Sigma-

Aldrich) and immediately analyzed under High Content Micro-Imaging

Acquisition and Screening System (Molecular Devices, Sunnyvale, CA,

USA). All parameters such as process count, branching, length, mature

neuron, and synapse density were analyzed by MetaXpress application

software (Molecular Devices).

MTT assay

After treatment with different duration of the oligomeric Aβ42 and

NaHS, cell viability was evaluated by using the MTT assay, which

measures the ability of metabolic active cells to form formazan through

cleavage of the tetrazolium ring of MTT (Agostinho and Oliveira, 2003).

Cells were incubated with MTT (0.5 mg/mL) for 4 h at 37℃. The blue

formazan crystals formed were dissolved in an equal volume of DMSO

and quantified spectrophotometrically by measuring the absorbance at

570 nm using a microplater spectrophotometer (uQuant, BioTek

Instruments Inc., Winooski, VT, USA.).

19

Establishment of the stress model

The stress model was established as previously described (Li et al.,

2006). As depicted in Fig. 1C, the 3×Tg-AD mice (n = 60) were

acclimatized in their home cages on days 1-5 and were handled once a

day during this 5-day habituation period. Handling consisted of holding

the animal in gloved hands for 2 min. After adaptation, mice received

either shocks (a total of 15 intermittent inescapable electric foot shocks of

0.8 mA intensity, 10 sec interval, and 10 sec duration; n = 30) or no

shocks (n = 30) on days 6 and 7, respectively. Prior to shock treatment,

the mice were allowed a 10-sec adaptation period in the shock box (the

dark compartment of the light-dark transition test box). The non-stress

groups received the same treatment, but with the shock mechanism

inactivated. The mice were re-exposed to the same chamber but without

foot shock treatment on days 8, 13, 20, and 31 (SR 1, 2, 3, and 4

respectively). After each situation reminders (SRs), a blood sample was

collected from each mouse and analyzed in order to measure the

corticosterone and H2S level. After SR3, mouse behavior was evaluated

by open field test, EPM, light–dark transition, and MWM on days 21-30,

respectively. Twenty-four hr after SR4 (conducted on day 31), the mice

were sacrificed for western blot, ELISA, and IHC analyses.

Open field test

Anxiety of the mice were assessed in a white open field box (30 cm ×

30 cm × 30 cm). A camera mounted on the ceiling above the chamber

connected to an automated video tracking system (EthoVision, Nodulus

20

Information Technology, Wageningen, Netherlands) was used to collect

and assess data in the absence of an observer for 5 min. The mouse was

placed in center, and the percentage of time spent in the central zone of

the field was measured as an anxiety level.

Light-dark transition test

The apparatus, modified from a previous study (Costall et al., 1989),

consisted of a Plexiglas chamber subdivided into two compartments, the

dark compartment (30 × 30 × 35 cm high)for the foot shocks, and a light

compartment (45 × 30 × 35 cm high; with a 60 W white bulb). The

compartments were connected by a small divider (50 × 50 mm). On day

21, each animal was placed in the light compartment facing the wall

opposite the divider. The latency before the first entry into the dark

compartment, the time spent in the dark compartment, and the numbers of

transitions were assessed for 5 min.

Elevated Plus-maze (EPM)

The elevated plus maze apparatus consisted of four arms (30 × 5 cm)

elevated 50 cm above floor level. Two of the arms contained 15 cm-high

walls (enclosed arms) and the other two none (open arms). Each mouse

was placed in the middle section facing an open arm and left to explore

the maze for a single 5 min session with the experimenter out of view.

After each trial, the floor was cleaned with 70% and 30% ethanol,

sequentially. Entries and duration in both arms were measured by a video-

camera and analyzed with the same video-tracking system as the open

21

field test.

Morris water maze (MWM) task

During conventional MWM training, an escape platform (10 cm in

diameter) made of white plastic, with a grooved surface for better grip,

was submerged 1.0 cm underneath the water level. Cues of various types

provided distal landmarks in the testing area of the room. The swimming

path of the mouse during each trial was recorded by a video camera

suspended 2.5 m above the center of the pool and connected to a video

tracking system (EthoVision; Nodulus). On the day prior to spatial

training, all mice underwent pre-training in order to assess their

swimming ability and to acclimatize them to the pool. In the three 60-sec

pre-training trials, the mouse was released into the water facing the wall

of the pool from semi-randomly-chosen cardinal compass points. After

three trials of acclimatization, each mouse was placed on the invisible

platform located at the center of the target quadrant and allowed to stay

there for 20 sec. The mice were given a 4-day training session consisting

of four 60-sec training trials (inter-trial interval: 20-30 min) per day. The

hidden platform was always placed at the same location of the pool

(Northeast quadrant as the target quadrant) throughout the training period.

During each trial, from semi-randomly chosen cardinal compass points,

the mouse was released into the water facing the pool wall. After

climbing onto the platform, the mouse was allowed to rest on it for 20

sec. If the mouse failed to swim to the platform within 60 sec or stay on it

for 20 sec, it would be placed on the platform by an experimenter.

22

Twenty-four hr after the last training trial, all mice were given three

testing trials to assess the time taken to climb onto the hidden platform.

Two and forty-eight hours after the last testing trial, all mice were given

two probe trials to evaluate the retrieval of the short- and long- term

spatial reference memory for the platform.

Immunohistochemistry (IHC)

Mice were anesthetized and transcardially perfused with 0.9% NaCl,

followed by 4% PFA in phosphate buffered saline (PBS). Mouse brains

were removed, post-fixed with 4% PFA for 4 hr, dehydrated by 10%

sucrose for 1 hr, 20% sucrose for 2 hr and 30% sucrose in PBS overnight

until sedimentation. The brains were preserved at -80°C until continuous

serial cryostat sectioning into 30 μm for immunostaining. Specific

primary and secondary antibodies used were listed in Table 2. In brief,

free-floating sections were washed with PBS for three times (10

min/wash). Nonspecific epitopes were then blocked by incubation in 3%

normal horse/goat/rabbit serum and 0.1% triton X-100 in PBS for 1 hr.

Sections were incubated in primary antibodies overnight at room

temperature, washed with PBS, and incubated with secondary antibodies

(1:200 dilution in blocking solution, Vector Laboratories, Burlingame

CA, USA) for 1 hr, then incubated in an avidin-biotin complex for 1 hr at

room temperature. The reaction was developed using a diaminobenzidine

(DAB) kit (Vector Laboratories). All sections were mounted on gelatin-

coated slides and cover-slipped for light microscopic observation. Signal

of positive staining neuron in specific area was first selected as standard

23

signal, and then the cell numbers of staining positive were counted by

digital image analysis software (Image Pro Plus, Media Cybernetics,

Washington, MD, USA). Pixel counts were taken as the average from

three adjacent sections per animal.

Western blot analysis

Proteins were extracted from the whole hippocampus of the mice (n

=3-5 per group). The protein concentration was determined using a

bicinchoninic acid (BCA) protein assay kit (Thermo, Rockford, IL,

USA). Proteins (25 μg) were separated by SDS-PAGE and transferred to

PVDF membranes. After transferring to PVDF membranes, the blots

were probed with various primary antibodies as listed in Table 3. The

same blot was probed for a housekeeping protein β-actin to serve as a

loading control. Secondary antibodies conjugated to anti-rabbit IgG HRP-

linked antibody (1:10000, Amersham Pharmacia Biotech, Piscataway, NJ,

USA) and anti-mouse IgG HRP-linked antibody (1:10000, Amersham)

were used. The specific antibody–antigen complex was detected by an

enhanced chemiluminescence detection system (Amersham). Quantitation

was performed using LAS-4000 chemiluminescence detection system

(Fujifilm, Tokyo, Japan), and target protein density was normalized to β-

actin internal control.

Enzyme-linked immunosorbent assay (ELISA) analysis

Blood was collected from facial vein by lancet after SRs. Blood was

mixed with heparin (20 units/ml) and centrifuged for 20 min, 4℃ at

24

2,000 × g. The supernatant was collected as plasma sample and store at -

80℃ until use. The levels of glutathione (GSH), IL-6 and corticosterone

in the plasma were measured using Glutathione assay kit (Cayman

Chemical, Ann Arbor, MI, USA), IL-6 ELISA kit (R&D Systems,

Minneapolis, MN, USA), and corticosterone competitive ELISA kit

(AssayPro system, GENTAUR, St. Charles, MO, USA). These assays

were following the manufacturer’s instructions.

Measurement of Plasma H2S

The collection of plasma was conducted as for ELISA. The tube was

filled with nitrogen gas and sealed with parafilm. Plasma (75 μl) was

mixed with 250 μl 1% Zn acetate (Sigma-Aldrich) and 450 μl double

distilled water for 10 min at room temperature. 250 μl 10%

Trichloroacetic acid (TCA; J.T. Baker, Center Valley, PA, USA) was then

added, centrifuged for 10 min, 4℃ at 14000 × g, and the clear

supernatant was collected and mixed with 133 μl 20 mM N,N-dimethyl-

p-phenylenediamine sulfate (Acros organics, Fair Lawn, NJ, USA) in 7.2

M HCl (Sigma-Aldrich) and 133 μl 30 mM FeCl3 (Sigma-Aldrich) in 1.2

M HCl. After 20 min, absorbance at 670 nm was measured with a

microplate reader (Multiskan GO, Thermo). The H2S concentration was

calculated against the calibration curve of the standard H2S solutions,

obtained by using 30% of the NaHS solution of various concentrations. It

was suggested that 30% of NaHS dissolved in water will be released as

HS−, which subsequently forms H2S with H+ (Kimura, 2000).

25

Aβ42 and Aβ40 ELISA assay

Levels of Aβ40 and Aβ42 in mouse hippocampus were measured by

ELISA (n = 5 per group). The whole hippocampal tissue was Dounce-

homogenized in 5 M guanidine hydrochloride (Sigma-Aldrich) and then

rocked gently at 4°C overnight. Samples were diluted with reaction buffer

containing protein inhibitors and 4-(2-Aminoethyl) benzenesulfonyl

fluoride hydrochloride (AEBSF, Calbiochem) to prevent degradation of

Aβ and centrifuged at 16,000 × g for 20 min at 4℃. After diluting the

supernatants with PBS, protein concentrations were determined by BCA

assay (Pierce), and levels of Aβ42 and Aβ40 were determined using

ELISA kits (Biosource International, Camarillo, CA, USA) according to

the manufacturer's protocol.

Statistical analysis

Data were analyzed by two-way analysis of variance (ANOVA) by

group (stress and non-stress) and administration (vehicle and NaHS),

followed by post hoc LSD analysis in order to compare the effects of all

treatments. In addition, the light-dark transition test and western blotting

results were analyzed by nonparametric multiple independent samples

testing. Results are expressed as mean ± S.E.M. Differences were

considered statistically significant if p < 0.05.

26

Results

Establishment of mouse hippocampal primary neuronal culture.

Hippocampal cultures are typically made from late-stage embryonic

tissue which contains fewer glial cells than those in mature brain tissues

(Banker and Cowan, 1977). Therefore, embryonic day 16-18 (E16-18)

embryos from C57BL/6J pregnant mice were used to isolate

hippocampus to identify suitable cultivating timing of culture. During the

in vitro culture, we utilized ICC staining to characterize the survival of

mature neurons and neuronal morphology (neuritic length, process, and

branch) at 6 days in vitro (DIV 6), 9 days in vitro (DIV 9), and 12 days in

vitro (DIV 12). From the results of ICC staining of cells, the different

cultivating timing significantly increased the number of total cells (F (2,

17) = 161.56, p < 0.001; Fig. 2A & B), glia cells (F (2, 17) = 143.28, p <

0.001; Fig. 2A & C), and decreased mature neurons (F (2, 17) = 57.56, p

< 0.001; Fig. 2A & D). However, there was no significant difference on

the neuritic length, processes, and branches at DIV 6, 9, and 12 (p > 0.05,

Fig. 2A, E, F, & G). From the post hoc analysis, there was no significant

difference on mature neurons and glia cells at DIV 9 as compared to DIV

12 (p > 0.05, Fig. 2A, C, & D). Therefore, DIV 9 was selected as a time

point for treatment.

In addition, previous study shows that AraC, a pyrimidine

antimetabolite, induced cytotoxicity of astrocytes (Mao and Wang, 2001a,

b). Therefore, AraC was applied in the mouse hippocampal primary

culture to reduce the overproliferation of astrocytes in the primary

culture. From the results of ICC staining in DIV 9, we found that AraC

27

significantly induced decreasing of total cells (F (2, 17) = 56.72; p <

0.001; Fig. 3A & B), astrocytes (F (2, 17) = 43.600, p < 0.001; Fig. 3A &

C), and increasing of mature neurons (F (2, 17) = 82.09; p < 0.001; Fig.

3A & D). However, there was no significant difference in neuritic length,

processes, and branches with the treatment of AraC (p >0.05; Fig. 3A, E,

F & G). From the post hoc analysis, the processes were significantly

increased in 2 μM AraC when compared to 0 μM AraC (p < 0.05; Fig. 3A

& F). From above results, the 2 μM AraC might be an optimized dose to

inhibit astrocytogenesis in the hippocampal primary neuronal culture.

Establishment of a mouse hippocampal primary neuronal culture

with oligomeric Aβ42 treatment

In addition, the platform of the hippocampal primary neuronal culture

treated with oligomeric Aβ42 was established in the study. We found that

the cell death level was significantly increased in the treatment of

oligomeric Aβ42 (F (1, 143) = 16.26, p < 0.0001; Fig. 4A), different

cultivating timing (F (5, 143) = 3.13, p < 0.05; Fig. 4A), and interaction

between the oligomeric Aβ42 and different cultivating timing (F (5, 143)

= 2.29, p < 0.05; Fig. 4A). From the post hoc comparison, the cell death

level was significantly increased after 1 hr (p < 0.05), 6 hr (p < 0.05), and

12 hr (p < 0.01). In addition, the level of LDH released in medium was

not changed in the cells after oligomeric Aβ42 treatment as compared to

vehicle group (p > 0.05; Fig. 4B). However, the level of LDH was

significantly increased in the different cultivating timing (F (5, 41) =

28.76, p < 0.0001; Fig. 4B). In addition, there was no significant

28

interaction in the oligomeric Aβ42 × different cultivating timing (p >

0.05; Fig. 4B). Furthermore, from the post hoc analysis, the level of LDH

released in the medium was significantly increased in the oligomeric

Aβ42 treatment as compared to the vehicle treatment at 1 hr (p < 0.05;

Fig. 4B).

From the quantification of ICC analysis, we found the total cell

numbers (F (1, 59) = 37.42, p < 0.0001; Fig. 5A & B), mature neurons (F

(1, 59) = 43.68, p < 0.0001; Fig. 5A & C), neuritic length (F (1, 59) =

78.56, p < 0.0001; Fig. 5A & D), processes (F (1, 59) = 51.66, p <

0.0001; Fig. 5A & E), branches (F (1, 59) = 57.57, p < 0.0001; Fig. 5A &

F), and synapse density (F (1, 59) = 45.07, p < 0.0001; Fig. 5A & G)

were significantly decreased in the oligomeric Aβ42-treated group as

compared to vehicle-treated group. In addition, the total cell numbers (F

(4, 59) =7.50, p < 0.0001; Fig. 5A & B), mature neurons (F (4, 59) =

14.29, p < 0.0001; Fig. 5A & C), neuritic length (F (4, 59) = 359.60, p <

0.0001; Fig. 5A & D), processes (F (4, 59) = 228.62, p < 0.0001; Fig. 5A

& E), branches (F (4, 59) = 895.44, p < 0.0001; Fig. 5A & F), and

synapse density (F (4, 59) = 390.53, p < 0.0001; Fig. 5A & G) had

significant differences among the different cultivating timing. In addition,

there was significant interaction between the oligomeric Aβ42 × different

cultivating timing in the total cell numbers (F (4, 59) = 4.21, p < 0.01;

Fig. 5A & B), the percentage of mature neurons (F (4, 59) = 6.64, p <

0.0001; Fig. 5A & C), neuritic length (F (4, 59) = 10.33, p < 0.0001; Fig.

5A & D), processes (F (4, 59) = 5.18, p < 0.01; Fig. 5A & E), (F (4, 59) =

7.83, p < 0.0001; Fig. 5A & F), and synapse density (F (4, 59) = 4.72, p <

29

0.01; Fig. 5A & G). From the results of the post hoc comparison, the total

cell and mature neurons were significantly decreased after the treatment

of oligomeric Aβ42 for 1 hr. However, neuritic length, process, branch,

and synapse density were significant decreased in the treatment of

oligomeric Aβ42 for 0.5 hr. Furthermore, after 6 hr cultivating timing,

neuritic length (F (4, 29) = 152.47, p < 0.01, Fig. 5A & D), processes (F

(4, 29) = 81.60, p < 0.01; Fig. 5A & E), and branches (F (4, 29) = 812.23,

p < 0.01; Fig. 5A & F) were significantly decreased in serum-free culture

medium. Therefore, these results suggest that treatment of oligomeric

Aβ42 for 1 hr was a proper timing for the following study.

The effects of NaHS in the oligomeric Aβ42 treated hippocampal

primary neuronal culture

In order to evaluate the effect of the NaHS in the oligomeric Aβ42

treated-hippocampal primary neuronal culture, we applied NaHS in

different doses, and timing (pre-treatment, post-treatment or co-

treatment) with the treatment of oligomeric Aβ42. Under the pretreatment

of NaHS, the cell viability (F (1, 29) = 22.57, p < 0.0001; Fig. 6A & B),

total cell numbers (F (1, 57) = 9.20, p < 0.01; Fig. 6A & C), the

percentage of mature neurons (F (1, 57) = 33.94, p < 0.0001; Fig. 6A &

D), neuritic length (F (1, 57) = 18.01, p < 0.0001; Fig. 6A & E),

processes (F (1, 57) = 48.03, p < 0.0001; Fig. 6A & F), and branches (F

(1, 57) = 11.61, p < 0.01; Fig. 6A & G) were significantly decreased in

the oligomeric Aβ42 as compared to vehicle treatment. However, the cell

viability (F (4, 29) = 12.10, p < 0.0001; Fig. 6A & B), neuritic length (F

30

(4, 57) = 19.42, p < 0.0001; Fig. 6A & E), processes (F (4, 57) = 25.64, p

< 0.0001; Fig. 6A & F), and branches (F (4, 57) = 21.00, p < 0.001; Fig.

6A & G) were significantly increased in the NaHS group as compared to

vehicle group. In addition, there was significant interaction between the

oligomeric Aβ42 and NaHS in the cell viability (F (4, 29) = 5.76, p <

0.01; Fig. 6A & B). Furthermore, the pretreatment of NaHS in the

concentration of 50 μM significantly increased the cell viability (p <

0.001; Fig. 6A & B), total cell (p < 0.01; Fig. 6A & C), neuritic length (p

< 0.05; Fig. 6A & E), processes (p < 0.001; Fig. 6A & F), and branches

(p < 0.05; Fig. 6A & G) against oligomeric Aβ42-induced neurotoxicity.

These results suggest that the pretreatment of NaHS was better in 50 μM

than in other dose against oligomeric Aβ42-induced neurotoxicity.

For the co-treatment of NaHS, the cell viability (F (1, 29) = 29.49, p

< 0.0001; Fig. 7A & B), total cell numbers (F (1, 59) = 11.16, p < 0.01;

Fig. 7A & C), the percentage of mature neurons (F (1, 59) = 47.77, p <

0.0001; Fig. 7A & D), neuritic length (F (1, 59) = 15.18, p < 0.001; Fig.

7A & E), processes (F (1, 59) = 10.82, p < 0.01; Fig. 7A & F), and

branches (F (1, 59) = 4.68, p < 0.05; Fig. 7A & G) were significantly

decreased in the oligomeric Aβ42 group as compared to vehicle group.

The total cell numbers (F (4, 59) = 5.31, p < 0.01; Fig. 7A & C), neuritic

length (F (4, 59) = 4.10, p < 0.01; Fig. 7A & E), processes (F (4, 59) =

20.85, p < 0.0001; Fig. 7A & F), and branches (F (4, 59) = 6.14, p <

0.001; Fig. 7A & G) were significantly increased in the NaHS group as

compared to vehicle group. In addition, there was significant interaction

between the oligomeric Aβ42 and NaHS in the cell viability (F (4, 29) =

31

10.60, p < 0.0001; Fig. 7A & B), and total cell numbers (F (4, 59) = 3.75,

p < 0.01; Fig. 7A & C). Furthermore, the co-treatment of NaHS in the

concentration of 50 μM significantly increased the cell viability (p <

0.001; Fig. 7A & B), total cell (p < 0.01; Fig. 7A & C), neuritic length (p

< 0.05; Fig. 7A & E), processes (p < 0.001; Fig. 7A & F), and branches

(p < 0.05; Fig. 7A & G) against oligomeric Aβ42-induced neurotoxicity.

For the post-treatment of NaHS, the cell viability (F (1, 29) = 4.73, p <

0.05; Fig. 8A & B), total cell numbers (F (1, 57) = 4.24, p < 0.05; Fig. 8A

& C), the percentage of mature neurons (F (1, 57) = 5.80, p < 0.05; Fig.

8A & D), neuritic length (F (1, 57) = 11.98, p < 0.01; Fig. 8A & E),

processes (F (1, 57) = 21.02, p < 0.0001; Fig. 8A & F), and branches (F

(1, 57) = 28.58, p < 0.0001; Fig. 8A & G) were significantly decreased in

the oligomeric Aβ42 group as compared to vehicle group. In addition, the

cell viability (F (4, 29) = 3.80, p < 0.05; Fig. 8A & B) and processes (F

(4, 57) = 3.55, p < 0.05; Fig. 8A & F) were significantly increased in the

NaHS group as compared to vehicle group. Significant interaction was

also identified between the oligomeric Aβ42 and NaHS in the cell

viability (F (4, 29) = 4.73, p < 0.01; Fig. 8A & B), and processes (F (4,

57) = 4.65, p < 0.01; Fig. 8A & F). Furthermore, the post-treatment of

NaHS in the concentration of 10 μM significantly increased the cell

viability (p < 0.01; Fig. 8A & B), total cell (p < 0.05; Fig. 8A & C), the

percentage of mature neurons (p < 0.05; Fig. 8A & D), neuritic length (p

< 0.01; Fig. 8A & E), process (p < 0.01; Fig. 8A & F), and branch (p <

0.01; Fig. 8A & G) against oligomeric Aβ42-induced neurotoxicity.

These results suggest that the 50 μM of pre-/co-treatment or 10 μM

32

post-treatment of NaHS could be effective against oligomeric Aβ42-

induced neurotoxicity.

Foot-shock induced mild stress associated with anxiety, but no effect

on levels of corticosterone and CRF in 3×Tg-AD mice

Recent evidence shows that stress experiences in young adults might

accelerate the cognitive loss in AD mice (Grigoryan et al., 2014).

Therefore, in the study, 3×Tg-AD mice (6 month old) received foot-shock

(a total of 15 intermittent inescapable electric foot-shocks of 0.8 mA

intensity) for 2 days associated with SRs in order to accelerate the

progression of AD. From the results of light-dark transition test, we found

that mice had been previously exposed to inescapable foot-shock

stimulation showed increased latency from the light to the dark

compartment (F (1, 41) = 17.15, p < 0.001; Fig. 9A), and decreased the

time spent in the dark compartment (F (1, 41) = 21.08, p < 0.001; Fig.

9B). In addition, inescapable foot-shock stimulation notably decreased

the frequency of dark-light transition of the mice (p > 0.05; Fig. 9C).

NaHS treatment didn’t affect the latency from the light to the dark

compartment (p > 0.05; Fig. 9A), the time spent in the dark compartment

(p > 0.05; Fig. 9B), and the frequency of dark-light transition (p > 0.05;

Fig. 9C). Furthermore, inescapable foot-shock stimulation and NaHS

treatment did not increase the mouse duration in the central zone during

open field test (p > 0.05; Fig. 9D) and ratio of open arms to closed arms

in EPM (p > 0.05; Fig. 9E). The levels of corticosterone in mouse plasma

were slightly decreased by stress on SR1, SR3, and SR4 (p > 0.05; Fig.

33

10A, C & D). In addition, the level of corticosterone was significantly

decreased in stress group with saline as compared to non-stress group

with saline on SR2. (p < 0.01; Fig. 10B). However, the administration of

NaHS in non-stress group mice significantly decreased the level of

corticosterone in plasma from SR1 to SR3 (p < 0.05, Fig. 10A; p < 0.01,

Fig. 10B; p < 0.01, Fig. 10C), but not extended to SR4 (Fig. 10D).

Moreover, the level of corticotropin-releasing factor (CRF), a peptide

hormone and neurotransmitter involved in the stress response, was also

not affected by stress (p > 0.05; Fig. 10E & F); however, CRF was

significantly decreased after NaHS administration (F (1, 11) = 6.43, p <

0.05; Fig. 10E & F). Therefore, the inescapable foot-shock only induced

mild stress and without increasing the levels of corticosterone and CRF in

3×Tg-AD mice.

Foot-shock decreased the concentration of H2S, and NaHS increased

the level of H2S in 3×Tg-AD mice

Stress induced by foot-shock significantly decreased the levels of

H2S in mouse plasma on SR2 (p < 0.05; Fig. 11B), SR3 (p < 0.001; Fig.

11C), and SR4 (p < 0.001; Fig. 11D). However, the administration of

NaHS in stress group significantly increased the level of H2S on SR2 (p <

0.05; Fig. 11B) and SR3 (p < 0.001; Fig. 11C). Furthermore, the

administration of NaHS in non-stress group also significantly increased

the level of H2S in plasma from SR3 to SR4 (p < 0.001, Fig. 11C; p <

0.001, Fig. 11D).

NaHS prevented cognitive dysfunction in 3×Tg-AD mice with mild

34

stress

During the training phase of MWM, mice with mild stress increased

the escape latencies onto the platform (F (1, 33) = 9.83, p < 0.01; Fig.

12A), however, NaHS treatment significantly decreased the escape

latencies as compared to vehicle treatment (F (1, 33) = 10.70, p < 0.01;

Fig. 12A). In the testing phase, mild stress significantly increased the

escape latencies onto the platform (F (1, 33) = 10.12, p < 0.01; Fig. 12B),

NaHS treatment also significantly decreased the latencies in stress group

(p < 0.001; Fig. 12B). In addition, NaHS treatment improved the short-

term memory (p < 0.01; Fig. 12C), not long-term memory in stress group

(p > 0.05; Fig. 12D). Therefore, mild stress induced the impairment of

spatial learning acquisition, however, the administration of NaHS

prevented the cognitive dysfunction in the 3×Tg-AD mice with mild

stress.

NaHS decreased PERK/eIF2α pathway associated with decreasing

BACE1, Aβ level in 3×Tg-AD mice with mild stress

Several reports indicated PERK pathway was involved in AD

pathogenesis (Salminen et al., 2009, Flight, 2013). In the study, mild

stress had no significant effect on the level of p-PERK/PERK ratio (p >

0.05; Fig. 13A & B). However, mild stress significantly increased the

level of p-eIF2α/eIF2α ratio (p > 0.05; Fig. 13A & C). In addition, the

administration of NaHS decreased the level of p-PERK/PERK ratio (F (1,

12) = 21.27, p < 0.01; Fig. 13A & B) and p-eIF2α/eIF2α ratio in non-

stress group (p < 0.01; Fig. 13A & C) and stress group (p < 0.5; Fig. 13A

35

& C). Furthermore, mild stress significantly increased the level of

BACE1 (F (1, 11) = 14.06, p < 0.01; Fig. 13A & D), and the

administration of NaHS decreased the level of BACE1 (F (1, 11) = 8.94,

p < 0.5; Fig. 13A & D).

For the level of Aβ analysis by ELISA, mild stress significantly

increased the concentration of Aβ40 (F (1, 13) = 19.97, p < 0.01; Fig.

14A) and Aβ42 (F (1, 11) = 8.09, p < 0.05; Fig. 14B) in the hippocampus,

however, the administration of NaHS significantly decreased the

concentration of Aβ40 (F (1, 13) = 80.40, p < 0.001; Fig. 14A) and Aβ42

in the hippocampus (F (1, 11) = 35.54, p < 0.001; Fig. 14B). In IHC

analysis, the numbers of Aβ40 positive staining cells were counted in the

CA1, CA3, and DG subregions of the hippocampus. Mild stress

significantly increased the Aβ40 positive cells in CA1 (F (1, 21) = 8.95, p

< 0.01; Fig. 14C & D), CA3 (F (1, 21) = 10.51, p < 0.01; Fig. 14C & E),

but not in DG (p > 0.05; Fig. 14C & F), and the administration of NaHS

significantly decreased Aβ40 positive staining cells in CA1 (F (1, 21) =

18.17, p < 0.001; Fig. 14C & D), CA3 (F (1, 21) = 59.69, p < 0.001; Fig.

14C & E), and DG (F (1, 21) = 37.17, p < 0.001; Fig. 14C & F). In

addition, mild stress significantly increased the Aβ42 positive cells in

CA1 (F (1, 26) = 14. 60, p < 0.001; Fig. 14C & G), CA3 (p < 0.05; Fig.

14C & H) and DG subregions of the hippocampus (F (1, 26) = 31.55, p <

0.001; Fig. 14C & I). Furthermore, the administration of the NaHS

significantly decreased the Aβ42 positive cells CA1 (F (1, 26) = 66.72, p

< 0.05; Fig. 14C & G), CA3 (F (1, 26) = 158.81, p < 0.001; Fig. 14C &

H), and DG subregions of the hippocampus (F (1, 26) = 13.10, p < 0.01;

36

Fig. 14C & I). These results show that the mild stress increased the Aβ

levels in the hippocampus, and the administration of NaHS reduced the

level of Aβ40 and Aβ42 in the hippocampus of the 3×Tg-AD mice.

NaHS induced anti-oxidation and anti-inflammation associated with

decreasing GSK3 kinase activation, Tau phosphorylation, and

activated glia in 3×Tg-AD mice with mild stress

For GSK3β analysis, we found that mild stress with saline treatment

significantly decreased the level of inactive pS9-GSK3β/GSK3β ratio

when compared to non-stress with saline treatment (p < 0.01; Fig.15A &

B). However, the NaHS increased the level of the pS9-GSK3β/GSK3β in

stress group (p < 0.05; Fig.15A & B). For GSK3α analysis, the mild

stress also didn’t change the level of the inactive pS21-GSK3α/GSK3α

ratio (p > 0.05; Fig. 15A & C), but the administration of NaHS increased

the level of the pS21-GSK3α/GSK3α ratio in stress group (p < 0.001;

Fig. 15A & C). For tau phosphorylation at different sites, mild stress

significantly increased Tau pS396 (F (1, 12) = 13.45, p < 0.05; Fig. 15D),

Tau pT231 (F (1, 11) = 28.61, p < 0.001; Fig. 15E), and Tau pS202 (F (1,

11) = 21.65, p < 0.01; Fig. 15F). However, the administration of NaHS

significantly decreased Tau pS396 (F (1, 12) = 6.30, p < 0.05; Fig. 15D),

Tau pS202 (F (1, 11) = 32.79, p < 0.001; Fig. 15F), and Tau pS262 (F (1,

13) = 6.49, p < 0.5; Fig. 15G). In addition, mild stress and the

administration of NaHS didn’t change the level of Tau pS404 and Tau

pT205 (data not shown). Therefore, the administration of NaHS reduced

Tau phosphorylation could be through decreasing GSK3 kinase

37

activation.

In addition, mild stress also significantly decreased the level of

MnSOD in hippocampus (F (1, 11) = 35.81, p < 0.001; Fig. 16A & B),

and the administration of NaHS significantly increased the level of

MnSOD (F (1, 11) =11.50, p < 0.01; Fig. 16A & B). Except oxidative

stress in hippocampus, the antioxidant component GSH concentration in

plasma was also increased in the NaHS treatment as compared with saline

treatment (F (1, 28) =10.39, p < 0.01; Fig. 16C).

Mild stress also increased the activated astrocyte in hippocampus (F

(1, 18) = 15.84, p < 0.01; Fig. 17A & B), and the administration of NaHS

significantly decreased the activated astrocyte (F (1, 18) = 11.13, p <

0.001; Fig. 17A & B).

Mild stress significantly increased the activated microglia in

hippocampus (p < 0.01; Fig. 17A & C), and the administration of NaHS

significantly decreased the activated microglia (F (1, 28) = 26.38, p <

0.0001; Fig. 17A & C). The pro-inflammatory cytokine IL-6

concentration in plasma was also significantly decreased in the NaHS

treatment as compared with saline treatment (F (1, 28) = 29.66, p < 0.001;

Fig. 17D). Therefore, these results showed that mild stress increased

oxidative stress and inflammation in the hippocampus, however, the

administration of NaHS rescued the responses.

NaHS increased the level of NR2A/NR2B ratio in 3×Tg-AD mice with

mild stress

Recent work has shown that the relative ratio of NR2A/NR2B was

38

affected by sensory experience and learning, as well as environmental

factors that impact learning such as sleep deprivation and stress (Baker

and Kim, 2002, Kart-Teke et al., 2006, Kopp et al., 2007). In the study,

mild stress did not change the level of NR2A/ NR2B ratio (F (1, 11) =

0.27, p > 0.05; Fig. 18A & B). However, the administration of NaHS

significantly increased the level of NR2A/NR2B ratio in 3×Tg-AD mice

with mild stress (p < 0.05; Fig. 18A & B).

NaHS induced anti-apoptosis and altered the number of cholinergic

and noradrenergic neurons in 3×Tg-AD mice

The levels of Bcl2 (an apoptosis inhibitor), Bax (an apoptosis

activator), and caspase 3 were measured by western blot (Fig. 19). Mild

stress significantly decreased the level of active caspase 3/total caspase 3

ratio (F (1, 12) = 26.63, p < 0.001; Fig. 19A &B), and the administration

of NaHS further largely reduced the level of active caspase 3/total

caspase 3 ratio in stress group (p < 0.05; Fig. 19A & B). In addition, mild

stress also significantly increased the level of Bcl2/Bax ratio (F (1, 11) =

13.87, p < 0.01; Fig. 19A & C), and the administration of NaHS further

largely enhanced the level of Bcl2/Bax ratio in stress group (p < 0.05;

Fig. 19A & C).

AD patients had a decline in the activity of choline acetyltransferase,

and acetylcholine esterase (Auld et al., 2002, Nyakas et al., 2011),

moreover, they also had a decline in acetylcholine release and associated

with cholinergic neuron loss (Davies and Maloney, 1976, Babic, 1999,

Kar et al., 2004). Norepinephrine is transmitter released from the

39

noradrenergic neuron, and the mean norepinephrine concentrations in AD

patients were significantly lower than those in control group (Zauner et

al., 2000, Heneka et al., 2010). Furthermore, the cholinergic neuron and

noradrenergic neuron have been a main focus of research in aging and

neural degradation such as AD.

In addition, the mild stress also significantly decreased the number of

cholinergic neurons (F (1, 19) = 8.88, p < 0.01; Fig. 20A & B), and the

administration of NaHS protected the cholinergic neurons (F (1, 19) =

22.84, p < 0.001; Fig. 20A & B) in the medial septum (MS), vertical

diagonal band of Broca (VDB), and horizontal diagonal band of Broca

(HDB) regions. However, mild stress did not induce significant difference

in the noradrenergic neurons (F (1, 22) = 12.44, p < 0.01; Fig. 20A & C),

and the administration of NaHS protect the noradrenergic neurons in the

noradrenergic neurons of locus coeruleus region (LC) region (F (1, 22) =

35.72, p < 0.001; Fig. 20A & C).

40

Discussion

In the study, the effects and molecular mechanisms of NaHS

administration were evaluated in hippocampal primary neuronal culture

and 3×Tg-AD mice with inescapable foot-shock stress.

From the results of in vitro, we have successful established the mouse

hippocampal primary culture and demonstrated that: 1) AraC could

reduce the percentage of astrocytes while neurons have normal

development of neuronal morphology ; 2) the oligomeric Aβ42 treatment

induced the cytotoxicity of culture in all of the aspects of neurons,

including neuron numbers, neuritic length, processes, and branches; and

3) treatment oligomeric Aβ42 for 1 hr is the optimal timing to evaluate

the effect of drug administration.

At first, AraC largely reduced the percentage of astrocyte and total

cell, but no effect in neurite outgrowth in the study. Previous evidence

also suggests that the treatment of AraC significantly reduced the

percentage of astrocytes only in hippocampal cultures, but increased the

percentage of apoptotic neurons in both hippocampal and cortical cultures

(Ahlemeyer and Baumgart-Vogt, 2005). Evidence further points out that

AraC in culture medium increased the purity and percentage of neurons

cultured (Wang et al., 2003). In addition, previous study shows that

neurite outgrowth was not affected in AraC-treated neuronal PC12 cells

(Chang and Brown, 1996). Therefore, AraC is a proper way to control the

astrocyte population with no significant damage for neuron growth.

A growing body of evidence suggests that soluble oligomers of

misfolded proteins/peptides played a major role in neurodegeneration and

41

which could be utilized as a screen platform to identify potential

compounds for neurodegenerative disease. In the study, hippocampal

primary neuronal culture was reduced neuritic length, processes,

branches, and synaptophysin treated with oligomeric Aβ42 for 0.5 hr.

However, the total cell and neuronal loss were induced after the treatment

of oligomeric Aβ42 for 1 hr. Furthermore, neuritic length, processes,

branches, and synaptophysin were reduced in serum-free culture medium

for 6 hr. Evidence shows that Aβ deposition is considered to cause

disruption of the neural network including progressive synaptic

degeneration and neuronal loss, which consequently results in cognitive

dysfunction and behavioral abnormalities in AD (Stepanichev et al.,

2004). Therefore, the hippocampal primary neuronal culture with 1-hr

oligomeric Aβ42-treatment could provide a fast screen platform for

potential therapeutic drugs in AD.

From the results of NaHS in vitro, we found that pre-, co-treatment of

NaHS in 50 M dose or post-treatment in 10 M dose increased total

cell, neuritic length, processes, and branches against the oligomeric

Aβ42-induced toxicity. Interestingly, further increase in the concentration

of NaHS did not increase the effect on neuronal morphology. Recent

evidence also suggests that the optimized dose of NaHS against Aβ42-

induced toxicity was 50 M (Zhu et al., 2014). Therefore, 50 M of

NaHS was used to elucidate the molecular mechanism.

From the results of the in vivo study, we suggest that 1) inescapable

foot-shock stimulation induced mild stress that impaired the spatial

learning acquisition, no effect on spatial memory; 2) mild stress increased

42

anti-apoptosis via increasing the level of Bcl2/Bax ratio; and 3) the

administration of NaHS prevented the cognitive dysfunction associated

with increasing of anti-oxidation and anti-inflammation via PERK/eIF2α

pathway and the level of NR2A/NR2B ratio in the 3×Tg-AD mice with

mild stress.

Chronic stress has been suggested as a risk factor for developing

AD. Therefore, in the study, 6-month-old 3×Tg-AD mice were received

foot-shock and associated with SRs in order to accelerate the progression

of AD. However, the stress only induced the impairment of spatial

learning acquisition associated with increasing of Aβ, tau protein

phosphorylation (at S396, T231, and S202), BACE1, and oxidative stress

in hippocampus and decreasing apoptosis in hippocampus, cholinergic

neurons in MS/DB region. In addition, the stress did not increase

peripheral corticosterone and CRF expression levels in hippocampus

during the experimental period. Furthermore, stress reduced the

concentration of H2S from SR2 to SR4. Evidence suggests that brain

hydrogen sulfide (H2S) synthesis was severely decreased in AD patients

(Giuliani et al., 2013). Previous evidences show that Aβ acumination and

Tau hyperphosphorylation had neurotoxicity and triggered oxidative

stress and inflammation (Feng and Wang, 2012, Ward et al., 2012).

Previous study also shows that chronic mild social stress on 3×Tg-AD

mice raised in corticosterone level, increased anxiety, elevated Aβ, and

decreased BDNF (Rothman et al., 2012). However, evidence suggests

that stress of unpaired flash light, foot-shock and a white noise session on

Wistar rats did not affect motor, exploratory activity, and plasma

43

corticosterone (Tishkina et al., 2012). In addition, study also suggests that

chronic combined unpredictable stress suppressed caspase-3 activity in

male Wistar rats (Tishkina et al., 2012). Report further points out that

social isolation did not induce remarkable effects on the genetically

determined AD-like symptoms resulting from differential susceptibility of

the 3×Tg-AD mouse line to environmental manipulations (Pietropaolo et

al., 2009). Furthermore, many reports also find that probe phase in MWM

was difficult to measure cognitive deficit in the 3×Tg-AD mice at 6

month old of age (Davis et al., 2013, Torres-Lista and Gimenez-Llort,

2013). Therefore, inescapable electric foot-shocks induced mild stress

and resulted in limited impacts on 3×Tg-AD mice.

Furthermore, we also found that the administration of NaHS in the

3×Tg-AD mice with mild stress ameliorated the impairment of spatial

learning and memory associated with increasing of anti-oxidation and

anti-inflammation, which could be through the PERK/eIF2α pathway. In

the study, the administration of NaHS reduced p-PERK/PERK ratio, p-

eIF2α/ eIF2α ratio, BACE1 level, Aβ level, Tau pS396, Tau pT231, Tau

pS202, activated microglia, activated astrocytes, activated caspase3 and

increasing MnSOD, GSH level in the serum, and levels of S9-

GSK3β/GSK3β, pS21- GSK3α/GSK3α, NR2A/NR2B, and Bcl2/Bax

ratio in the hippocampus of the 3×Tg-AD mice with mild stress.

Evidence suggests that increasing the level of p-PERK/PERK ratio might

activate GSK3 kinase and result in inflammation in AD (Salminen et al.,

2009). In addition, study also shows that oxidative stress increased the

expression of BACE1 via activation of the PERK/eIF2α pathway

44

(Mouton-Liger et al., 2012).

Therefore, we suggest that H2S might be a potential therapeutic

strategy to prevent the cognitive dysfunction of the AD.

45

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54

Tables & Figures

Table 1. Primary antibodies used in immunocytochemistry

Antibody Catalog Host species Titer Source

NeuN MAB377 Mouse 1:1000 Millipore, Temecula, CA, USA

MAP2 AB5622 Rabbit 1:1000 Millipore, Temecula, CA, USA

Nestin ab6142 Mouse 1:1000 Abcam, Cambridge CB4 0FL, UK

Synaptophysin ab14692 Rabbit 1:1000 Abcam, Cambridge CB4 0FL, UK

55

Table 2. Primary antibodies used in immunohistochemistry

Antibody Catalog Host species Titer Source

Choline

Acetyltransferase

AB143 Rabbit 1:500 Millipore, Temecula, CA, USA

Aβ40 44136 Rabbit 1:2000 Invitrogen, Camarillo, CA, USA

Aβ42 700254 Rabbit 1:500 Invitrogen, Camarillo, CA, USA

GFAP MAB360 Mouse 1:1000 Millipore, Temecula, CA, USA

Iba1 019-19741 Rabbit 1:1000 Wako, Osaka, Japan

Tau-pSer202 28017-025 Rabbit 1:1000 AnaSpec, San Jose, CA, USA

Serotonin MAB352 Rat 1:100 Millipore, Temecula, CA, USA

Tyrosine

Hydroxylase

AB152 Rabbit 1:1000 Millipore, Temecula, CA, USA

56

Table 3. Primary antibodies used in western blot

Antibody Catalog Host species Titer Size

(kDa)

Source

NMDA receptor

2A

AB1555 Rabbit 1:1000 180 Millipore, Temecula, CA, USA

NMDA receptor

2B

AB1557 Rabbit 1:1000 180 Millipore, Temecula, CA, USA

actin MAB1501 Mouse 1:1000 43 Millipore, Temecula, CA, USA

cleaved caspase-3 AB3623 Rabbit 1:1000 17/19 Millipore, Temecula, CA, USA

MnSOD 06-984 Rabbit 1:1000 24 Upstate, Lake Placid, NY, USA

GSK3β 1561-1 Rabbit 1:1000 47 Epitomics, Burlingame, California, USA

phospho-GSK3β 2435 Rabbit 1:1000 47 Epitomics, Burlingame, California, USA

GSK3α 9338 Rabbit 1:1000 51 Cell Signaling, Danvers, MA, USA

phospho-GSK3α 9316 Rabbit 1:1000 51 Cell Signaling, Danvers, MA, USA

BACE 5606 Rabbit 1:1000 60-70 Cell Signaling, Danvers, MA, USA

Tau-pS396 44752G Rabbit 1:1000 70 Invitrogen, Camarillo, CA, USA

Tau-pS202 28017-025 Rabbit 1:1000 52 AnaSpec, San Jose, CA, USA

Tau-pT205 44738G Rabbit 1:1000 70 Invitrogen, Camarillo, CA, USA

Tau-pT231 44746G Rabbit 1:1000 70 Invitrogen, Camarillo, CA, USA

Tau-pS404 44758G Rabbit 1:1000 80 Invitrogen, Camarillo, CA, USA

Tau-pS262 AB9656 Rabbit 1:1000 52 Millipore, Temecula, CA, USA

Tau 5 MAB361 Mouse 1:1000 45-68 Millipore, Temecula, CA, USA

iNOS 160862 Rabbit 1:1000 130 Cayman Chemical, MI, USA

CRF SC-1759 Mouse 1:1000 25 Santa Cruz Biotechnology, Delaware, CA,

USA

57

Bcl2 B3170 Mouse 1:1000 26 Sigma-Aldrich, St. Louis, MO, USA

Bax 556467 Mouse 1:1000 21-22 BD Pharmingen, San Diego, CA, USA

PERK 3192 Rabbit 1:1000 140 Cell Signaling, Danvers, MA, USA

phospho-PERK 3179 Rabbit 1:1000 170 Cell Signaling, Danvers, MA, USA

eIF2α 9722 Rabbit 1:1000 38 Cell Signaling, Danvers, MA, USA

phosphor- eIF2α 9721 Rabbit 1:1000 38 Cell Signaling, Danvers, MA, USA

58

59

A

C

B

C57BL/6E16-18 DIV9

Aβ1-42 oligomer (1 μM) vehicle

30 min 1 hr

1. MTT assay2. medium LDH3. ICC: NeuN, Nestin MAP2

12 hr6 hr

C57BL/6E16-18 DIV9

1 hr-30 min +30 min

1, 10, 50 & 100 μM NaHS

0 min

1. MTT assay2. ICC: NeuN, MAP2

and synaptophysin

Aβ1-42 oligomer (1 μM) vehicle

APP/PS1/Tau ♂(6 month )

NaHS (2.5 mg/Kg), twice a day

1. Open field test2. Light-Dark

transition test3. EPM

1.SR42.IHC3.WB4. ELISA

Day 1 6 7 8 13 20 21 22 23 24 31 32

MWMFoot-shock SR1 SR2 SR3

60

Fig. 1. Experimental timelines in this study. (A) Time course of

oligomeric Aβ42 treatment on hippocampal primary culture. (B) At DIV9,

different doses of NaHS (1, 10, 50 or 100 μM) were applied to the

hippocampal primary culture 30 min before (pre-treatment), after (post-

treatment), or at the same time (co-treatment) with oligomeric Aβ42. (C)

The timeline of the 6-month-old 3×TG-AD mice with inescapable electric

foot shocks, behavioral analyses, and pathologic analyses under NaHS or

vehicle treatment.

61

A

D

F

B

Days in vitro

6 9 12

Pro

cess (

%)

0

50

100

150

200

Days in vitro

6 9 12

Bra

nch

(%

)

0

50

100

150

200

Days in vitro

6 9 12

Neu

rite

len

gth

(%

)

0

50

100

150

200

Days in vitro

6 9 12

% o

f N

eu

N p

osit

ive c

ells

(rati

o t

o t

ota

l cells)

0

10

20

30

40

***

Days in vitro

6 9 12

To

tal

ce

lls

(1

x1

04

)

0

5

10

15

20

***

***

***

Days in vitro

6 9 12

% o

f g

lia c

ells

(rati

o t

o t

ota

l cells)

0

5

10

15

20

***

C

E

G

62

Fig. 2. The effects of different culture days in hippocampal primary

cells. (A) Immunocytochemistry staining of NeuN, MAP2, and DAPI

after culture for 6, 9, and 12 days. Scale bar: 100 μm. (B-G) Quantitation

of total cell number, mature neurons, astrocytes, neuritic length, processes

and branches. Data are expressed as means ± S.E.M. *, p < 0.05; **, p <

0.01; ***, p < 0.001.

63

AraC (M)

0 2 4

Ne

uri

te le

ng

th (

%)

0

50

100

150

200

AraC (M)

0 2 4

Pro

ce

ss

(%

)

0

50

100

150

200

*

AraC (M)

0 2 4

Bra

nc

h (

%)

0

50

100

150

200

AraC (M)

0 2 4

To

tal

ce

lls

(1

x1

04

)

0

2

4

6

8

10

***

AraC (M)

0 2 4

% o

f g

lia

ce

lls

(r

ati

o t

o t

ota

l c

ells

)

0

5

10

15

20

***

AraC (M)

0 2 4

% o

f N

eu

N p

os

itiv

e c

ells

(r

ati

o t

o t

ota

l c

ells

)

0

20

40

60

80

*****

A

D

F

B C

E

G

64

Fig. 3. The effects of AraC in hippocampal primary cells in DIV 9.

(A) Immunocytochemistry staining of NeuN, MAP2, and DAPI after

culture in DIV 9. Scale bar: 100 μm. (B-G) Quantitation of total cell

number, mature neurons, astrocytes, neuritic length, processes and

branches. Data are expressed as means ± S.E.M. *, p < 0.05; **, p < 0.01;

***, p < 0.001.

65

*

Time after treatment (hr)

0 0.5 1 6 12LD

H r

ele

ase

in

med

ium

(ra

tio

to

co

ntr

ol)

0

50

100

150

200Vehicle

A

*

Time after treatment (hr)

0 0.5 1 6 12

Cell

via

bil

ity

0

25

50

75

100

125 Vehicle

A

* *** ***

A B

66

Fig. 4. The cytotoxic effect oligomeric Aβ42 treatment on mouse

hippocampal primary culture. (A) MTT assay showed oligomeric Aβ42

decreased cell viability. (B) LDH release after oligomeric Aβ42

treatment. Data are expressed as means ± S.E.M. *, p < 0.05; **, p <

0.01; ***, p < 0.001.

67

Time after treatment (hours)

0 0.5 1 6 12

Pro

ces

s (

%)

0

25

50

75

100

125

150Vehicle

A

****

**

*

Time after treatment (hours)

0 0.5 1 6 12

To

tal

cell

s (

1x

104)

0.0

0.5

1.0

1.5

2.0 Vehicle

A

*** *****

Time after treatment (hours)

0 0.5 1 6 12

Bra

nc

h (

%)

0

25

50

75

100

125

150Vehicle

A

****

*

**

Time after treatment (hours)

0 0.5 1 6 12

% o

f N

eu

N p

osit

ive c

ells

(rati

o t

o t

ota

l cells)

0

20

40

60

80Vehicle

A

** ** ***

C

Time after treatment (hours)

0 0.5 1 6 12

Neu

rite

le

ng

th (

%)

0

20

40

60

80

100

120Vehicle

A

**

**

**

**

Time after treatment (hours)

0 0.5 1 6 12

Syn

ap

top

hys

in (

%)

0

20

40

60

80

100

120 Vehicle

A

**

****** **

A

B D

E F G

Aβ

Ve

hic

le

0.5 hr 1 hr 6 hr 12 hr

NeuN MAP2 DAPI

68

Fig. 5. The effects of oligomeric Aβ42 on primary culture analyzed by

immunocytochemical staining. (A) Staining of NeuN, MAP2, and DAPI

after oligomeric Aβ42 treatment for 0, 0.5, 1, 6, 12 hr. Scale bar: 100 μm.

(B-G) Quantitation of total cell number, mature neurons, neuritic length,

processes, and branches. Data are expressed as means ± S.E.M. *, p

< .05; **, p < 0.01; ***, p < 0.001.

69

Pro

cess (

%)

0

25

50

75

100

125

1500 M

1 M

10 M

50 M

100 M

Vehicle A

***

****

**

***

NaHS

% o

f N

eu

N p

osit

ive

cell

s

(ra

tio

to

to

tal c

ell

s)

0

10

20

30

40

500 M

1 M

10 M

50 M

100 M

Vehicle A

***

NaHS

Cell

via

bil

ity (

%)

0

25

50

75

100

125

150

0 M

1 M

10 M

50 M

100 M

Vehicle A

*

*

***

***NaHS

Vehicle Aβ

NaHS 0 μM 50 μM

A

B

D

To

tal

cell

s (

1x

104)

0.0

0.5

1.0

1.5

2.00 M

1 M

10 M

50 M

100 M

Vehicle A

**

**NaHS

Neu

rite

len

gth

(%

)

0

50

100

150

200

2500 M

1 M

10 M

50 M

100 M

Vehicle A

***

*

**

***

***

NaHS

F

C

E

NeuN MAP2 DAPI

0 μM 50 μM

Bra

nch

(%

)

0

25

50

75

100

125

1500 M

1 M

10 M

50 M

100 M

Vehicle A

*

*** **

***

*

NaHS

G

70

Fig. 6. Effect of NaHS pre-treatment on hippocampal primary

neuronal culture with oligomeric Aβ42. (A) Immunocytochemistry

staining with NeuN, MAP2, and DAPI. Scale bar: 100 μm. (B-G)

Quantitation of cell viability, total cell number, mature neurons, neuritic

length, processes, and branches. Data are expressed as means ± S.E.M. *,

p < 0.05; **, p < 0.01; ***, p < 0.001

71

NeuN MAP2 DAPI

Cell v

iab

ilit

y (

%)

0

25

50

75

100

125

150

0 M

1 M

10 M

50 M

100 M

Vehicle A

*

***

**

*** NaHS

% o

f N

eu

N p

osit

ive c

ells

(rati

o t

o t

ota

l cells)

0

10

20

30

40

50

0 M

1 M

10 M

50 M

100 M

Vehicle A

***NaHS

Pro

ces

s (

%)

0

100

200

300

400

500

0 M

1 M

10 M

50 M

100 M

Vehicle A

*****

***

** NaHS

B

F

Vehicle Aβ

NaHS 0 μM 50 μM

A

0 μM 50 μM

D

To

tal cells (

1x104)

0.0

0.5

1.0

1.5

2.0

0 M

1 M

10 M

50 M

100 M

Vehicle A

** **

** NaHS

Neu

rite

len

gth

(%

)

0

50

100

150

200

250

0 M

1 M

10 M

50 M

100 M

Vehicle A

*

*

***NaHS

Bra

nch

(ra

tio

to

veh

icle

co

ntr

ol)

0

100

200

300

400

500

0 M

1 M

10 M

50 M

100 M

Vehicle A

***

*NaHS

C

E

G

72

Fig. 7. Effects of NaHS co-treatment on hippocampal primary

neuronal culture with oligomeric Aβ42. (A) Immunocytochemistry

staining with NeuN, MAP2, and DAPI. Scale bar: 100 μm. (B-G)

Quantitation of cell viability, total cell number, mature neurons, neuritic

length, processes, and branches. Data are expressed as means ± S.E.M. *,

p < 0.05; **, p < 0.01; ***, p < 0.001.

73

Cell

via

bil

ity (

%)

0

25

50

75

100

125

150

0 M

1 M

10 M

50 M

100 M

Vehicle A

*

**

***

* NaHS

To

tal cells (

1x104)

0.0

0.5

1.0

1.5

2.0

Vehicle A

0 M

1 M

10 M

50 M

100 M

*

* NaHS

% o

f N

eu

N p

osit

ive c

ells

(rati

o t

o t

ota

l cells)

0

10

20

30

40

50

0 M

1 M

10 M

50 M

100 M

Vehicle A

*

*

NaHS

Neu

rite

le

ng

th (

%)

0

50

100

150

200

250

0 M

1 M

10 M

50 M

100 M

Vehicle A

**

***

NaHS

Pro

cess (

%)

0

25

50

75

100

125

150

0 M

1 M

10 M

50 M

100 M

Vehicle A

***

*** NaHS

Bra

nc

h (

%)

0

25

50

75

100

125

150

0 M

1 M

10 M

50 M

100 M

Vehicle A

**

*** NaHS

NeuN MAP2 DAPI

B C

D E

F G

Vehicle Aβ

NaHS 0 μM 10 μM

A

0 μM 10 μM

74

Fig. 8. Effects of NaHS post-treatment on hippocampal primary

neuronal culture with oligomeric Aβ42. (A) Immunocytochemistry

staining with NeuN, MAP2, and DAPI. Scale bar: 100 μm. (B-G)

Quantitation of cell viability, total cell number, mature neurons, neuritic

length, processes, and branches. Data are expressed as means ± S.E.M. *,

p < 0.05; **, p < 0.01; ***, p < 0.001.

75

Non-stress StressLate

ncy t

o D

ark

co

mp

art

men

t (s

eco

nd

s)

0

50

100

150

200

250

300Saline

NaHS

##

Non-stress Stress

Du

rati

on

in

cen

tral

zo

ne (

seco

nd

s)

0

50

100

150

200

250

300Saline

NaHS

Non-stress StressDu

rati

on

in

dark

co

mp

art

men

t (s

eco

nd

s)

0

50

100

150

200

250

300Saline

NaHS

#

Non-stress Stress

Rati

o o

f o

pe

n a

rms

to

clo

se

d a

rms

0

20

40

60

80Saline

NaHS

Non-stress Stress

Tim

e o

f tr

an

sit

ion

(co

un

ts)

0

3

6

9

12

15Saline

NaHS

A

C

B

D

E

76

Fig. 9. Characterization of the mouse anxiety-like behavior with

light-dark transition, open field test, and EPM tests. (A) The escape

latencies from the light compartment to the dark compartment were

increased by stress. (B) The time spent in the dark compartment was

decreased by stress. (C) The frequency of dark-light transition was not

affected by stress. (D) The duration in the central zone of open field test

was no affect by stress or NaHS treatment. (E) In EPM, the ratio of open

arms to closed arms was not changed by stress. Data are expressed as

means ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. saline; #, p <

0.05; ##, p < 0.01; ###, p < 0.001 vs. non-stress/saline.

77

SR1

Non-stress Stress

Co

rtic

os

tero

ne

(n

g/m

l)

0

20

40

60

80

100Saline

NaHS

*

SR2

Non-stress Stress

Co

rtic

os

tero

ne

(n

g/m

l)

0

20

40

60

80

100Saline

NaHS

** ##

SR3

Non-stress Stress

Co

rtic

os

tero

ne

(n

g/m

l)

0

20

40

60

80

100Saline

NaHS

**

SR4

Non-stress Stress

Co

rtic

os

tero

ne

(n

g/m

l)

0

20

40

60

80

100Saline

NaHS

Non-stress Stress

CR

F (

rati

o t

o a

cti

n)

0.0

0.5

1.0

1.5

2.0aline

NaHS

* *

A

C

B

D

E F

78

Fig. 10. Plasma corticosterone level and hippocampal CRF in the

stressed and non-stressed mice. The level of corticosterone in (A) SR1,

(B) SR2, (C) SR3, and (D) SR4 were not changed by stress. (E-F) The

levels of CRF were also not affected by stress. The administration of

NaHS decreased the CRF level. Data are expressed as means ± S.E.M. *,

p < 0.05; **, p < 0.01; ***, p < 0.001 vs. saline; #, p < 0.05; ##, p < 0.01;

###, p < 0.001 vs. non-stress/saline.

79

SR1

Non-stress Stress

Co

nc.

of

H2S

in

pla

sm

a (

M)

0

100

200

300

400Saline

NaHS

SR2

Non-stress Stress

Co

nc.

of

H2S

in

pla

sm

a (

M)

0

100

200

300

400Saline

NaHS

# *

SR3

Non-stress Stress

Co

nc.

of

H2S

in

pla

sm

a (

M)

0

100

200

300

400Saline

NaHS

###

***

***

SR4

Non-stress Stress

Co

nc.

of

H2S

in

pla

sm

a (

M)

0

100

200

300

400Saline

NaHS

***

###

A

C

B

D

80

Fig. 11. Plasma H2S level in the stressed mice treated with NaHS. The

level of H2S in (A) SR1, (B) SR2, (C) SR3, and (D) SR4 were decreased

by stress. The administration of NaHS increased the H2S level. Data are

expressed as means ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs.

saline; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 vs. non-stress/saline.

81

Training day

1 2 3 4

Escap

e late

ncy (

seco

nd

s)

010

20

30

40

50 Non-stress/ Saline

Non-stress/ NaHS

Stress/ Saline

Stress/ NaHS

##

***

Non-stress Stress

Du

rati

on

in

targ

et

reg

ion

(seco

nd

s)

0

10

20

30

40

50

60Saline

NaHS

**

2 hr probe

Non-stress Stress

Du

rati

on

in

targ

et

reg

ion

(seco

nd

s)

0

10

20

30

40

50

60Saline

NaHS

48 hr probe

DC

BA

Non-stress Stress

Escap

e late

ncy (

seco

nd

s)

0

10

20

30

40

50

60Saline

NaHS

***

##

82

Fig. 12. The effects of NaHS treatment and stress on mouse

performance in the Morris water maze task. During 4 training sessions

(A) and testing phase (B), stress increased escape latency, and NaHS

treatment decreased the escape latency onto the platform. After testing,

the probe trial was conducted 2 hr (C), and 48 hr (D) after the last testing

trial for assessment of short-term and long-term memory, respectively.

NaHS treatment improved the short-term memory, not long-term

memory. Data are expressed as means ± S.E.M. *, p < 0.05; **, p < 0.01;

***, p < 0.001 vs. saline; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 vs.

non-stress/saline.

83

Non-stress Stress

BA

CE

1 (

rati

o t

o a

cti

n)

0.0

0.5

1.0

1.5

2.0Saline

NaHS

#

*

A

Non-stress Stress

p-e

IF2

/ eIF

2a

0

1

2

3

4Saline

NaHS

#

** *

Non-stress Stress

p-P

ER

K/ P

ER

K

0.0

0.5

1.0

1.5

2.0Saline

NaHS

**

B

C

D

84

Fig. 13. Immunoblot analyses of PERK/eIF2α pathway and BACE1

protein expression in non-stressed and stressed mice treated with

saline or NaHS. (A) Results of western blot of the protein from the

mouse hippocampus. (B) Quantification of p-PERK/PERK level. (C)

Quantification of p-eIF2α/eIF2α level. (D) Quantification of BACE1

level. Data are expressed as means ± S.E.M. *, p < 0.05; **, p < 0.01;

***, p < 0.001 vs. saline; #, p < 0.05; ##, p < 0.01; ###, p < 0.001 vs.

non-stress/saline.

85

Non-stress StressCo

nc.

of

A4

0 i

n h

ipp

oc

am

pu

s (

pg

/ml)

0

20

40

60

80

100Saline

NaHS

* **

##

Non-stress Stress

A40 p

osit

ive c

ells in

CA

1 (

co

un

t)

0

50

100

150

200Saline

NaHS ##

*

**

Non-stress Stress

A40 p

osit

ive c

ells in

CA

3 (

co

un

t)

0

100

200

300

400Saline

NaHS

#

****

Non-stress Stress

A40 p

osit

ive c

ells in

DG

(co

un

t)

0

100

200

300

400

500Saline

NaHS

***

*

A B

Non-stress StressCo

nc. o

f A42 in

hip

po

cam

pu

s (

pg

/ml)

0

20

40

60

80Saline

NaHS

***

#

Non-stress Stress

A42 p

osit

ive c

ells in

CA

1 (

co

un

t)

0

20

40

60

80

100Saline

NaHS ##

***

Non-stress Stress

A42 p

osit

ive c

ells in

CA

3 (

co

un

t)

0

50

100

150

200Saline

NaHS

#

*** ***

Non-stress Stress

A42 p

osit

ive c

ells in

DG

(co

un

t)

0

50

100

150

200Saline

NaHS

###

**

D E F

G H I

Non-stress Stress

Saline NaHS Saline NaHS

Aβ

40

Aβ

42

C

86

Fig. 14. The levels of Aβ were analyzed in mouse hippocampus non-

stressed and stressed mice treated with saline or NaHS. (A) The

concentration of Aβ40 in hippocampus. (B) The concentration of Aβ42 in

hippocampus. (C) Immunohistochemistry staining of Aβ40 and Aβ42 in

hippocampal CA1, CA3, and DG. (D-F) Quantification of Aβ40 positive

cells in different regions of hippocampus. (G-I) Quantification of Aβ42

positive cells in different regions of hippocampus. Data are expressed as

means ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. saline; #, p <

0.05; ##, p < 0.01; ###, p < 0.001 vs. non-stress/saline.

87

Non-stress Stress

pS

9-G

SK

3/

GS

K3

0.0

0.5

1.0

1.5

2.0

2.5Saline

NaHS

##

*

Non-stress Stress

pS

21-G

SK

3

/ G

SK

3a

0.0

0.5

1.0

1.5

2.0Saline

NaHS

***

Non-stress Stress

Tau

pS

396/ T

au

5

0.0

0.5

1.0

1.5

2.0

2.5Saline

NaHS

#

*

Non-stress Stress

Tau

pT

231/ T

au

5

0.0

0.5

1.0

1.5

2.0Saline

NaHS

#

**

Non-stress Stress

Tau

pS

202

/ T

au

5

0

1

2

3

4Saline

NaHS

#

**

Non-stress Stress

Tau

pS

262

/ T

au

5

0.0

0.5

1.0

1.5

2.0

2.5Saline

NaHS

*

A B C

D E

F G

88

Fig. 15. Western blot analysis of the protein level of inactive form of

GSK3β, GSK3α, and Tau phosphorylation. (A) Results of western blot

of the proteins from the mouse hippocampus. (B) Quantification of pS9-

GSK3β/GSK3β ratio. (C) Quantification of pS21-GSK3α/GSK3α ratio.

(D) Quantification of Tau pS396 level. (E) Quantification of Tau pT231

level. (F) Quantification of Tau pS202 level. (G) Quantification of Tau

pS206 level. Data are expressed as means ± S.E.M. *, p < 0.05; **, p <

0.01; ***, p < 0.001 vs. saline; #, p < 0.05; ##, p < 0.01; ###, p < 0.001

vs. non-stress/saline.

89

Non-stress Stress

Mn

SO

D (

rati

o t

o a

cti

n)

0.0

0.5

1.0

1.5

2.0Saline

NaHS

#*

Non-stress Stress

Co

nc. o

f G

SH

in

pla

sm

a (

M)

0

5

10

15

20Saline

NaHS

**

A B C

90

Fig. 16. The anti-oxidative effects in non-stressed and stressed mice

treated with saline or NaHS. (A) Results of western blot of the proteins

from the mouse hippocampus. (B) Quantification of MnSOD level. (C)

Concentration of GSH in plasma. Data are expressed as means ± S.E.M.

*, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. saline; #, p < 0.05; ##, p <

0.01; ###, p < 0.001 vs. non-stress/saline.

91

Non-stress Stress

Co

nc. o

f IL

-6 in

pla

sm

a (

pg

/ml)

0

20

40

60

80Saline

NaHS

**

**

D

Non-stress Stress

As

tro

cyte

(c

ou

nt)

0

20

40

60

80Saline

NaHS

**

##

A

B

Non-stress Stress

Mic

rog

lia

(co

un

t)

0

20

40

60

80Saline

NaHS

***

##

C

GFA

PIb

a1Non-stress Stress

Saline NaHS Saline NaHS

100 μm

92

Fig. 17. The anti-inflammatory effects in non-stressed and stressed

mice treated with saline or NaHS. (A)IHC staining of astrocytes and

microglia in hippocampus. (B) Quantification of astrocytes in

hippocampus. (C) Quantification of microglia in hippocampus. (D)

Concentration of IL-6 in plasma. Data are expressed as means ± S.E.M.

*, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. saline; #, p < 0.05; ##, p <

0.01; ###, p < 0.001 vs. non-stress/saline.

93

Non-stress StressN

R2A

/ N

R2B

0.0

0.5

1.0

1.5

2.0Saline

NaHS *

A B

94

Fig. 18. The protein levels of NR2A and NR2B in non-stressed and

stressed mice treated with saline or NaHS. (A) Results of western blot

of the protein from the mouse hippocampus. (B) Quantification of

NR2A/NR2B ratio. Data are expressed as means ± S.E.M. *, p < 0.05; **,

p < 0.01; ***, p < 0.001 vs. saline; #, p < 0.05; ##, p < 0.01; ###, p <

0.001 vs. non-stress/saline.

95

Non-stress Stress

Bcl2

/ B

ax

0.0

0.5

1.0

1.5

2.0

2.5Saline

NaHS

#

*

A

Non-stress Stress

Ac

tive

cas

pa

se

3/

tota

l c

as

pa

se

3

0.0

0.5

1.0

1.5

2.0Saline

NaHS

#

*

B

C

96

Fig. 19. Caspase 3 and Bcl2/Bax in non-stressed and stressed mice

treated with saline or NaHS. (A) Results of western blot of the protein

from the mouse hippocampus. (B) Quantification of active caspase 3/total

caspase 3 ratio. (C) Quantification of Bcl2/Bax ratio. Data are expressed

as means ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. saline; #,

p < 0.05; ##, p < 0.01; ###, p < 0.001 vs. non-stress/saline.

97

Non-stress Stress

Ch

oli

ne

rgic

neu

ron

s (

co

un

t)

0

100

200

300

400Saline

NaHS

*

*

#

Non-stress Stress

No

rad

ren

erg

ic n

eu

ron

s (

co

un

t)

0

25

50

75

100

125

150Saline

NaHS

***

**

Non-stress Stress

Saline NaHS Saline NaHS

Ch

AT

TH

A

B C

98

Fig. 20. Cholinergic, and noradrenergic neurons in non-stressed and

stressed mice treated with saline or NaHS. (A) Immunohistochemistry

staining of ChAT in the MS/DB, and TH in the LC. (B) Quantification of

cholinergic neurons in the MS/DB. (C) Quantification of noradrenergic

neurons in the LC. Data are expressed as means ± S.E.M. *, p < 0.05; **,

p < 0.01; ***, p < 0.001 vs. saline; #, p < 0.05; ##, p < 0.01; ###, p <

0.001 vs. non-stress/saline.

99

Appendixes

Appendix 1. Phase-contract images of hippocampal neurons in different

stage. a) Hippocampal neurons extend a lamella after planting; b) there

were several minor neuritis undergoes growth; c) the minor neurites of

neurons grow continuously, becoming two to three times longer than the

other; and d) the dendrites have begun to grow and branch (Kaech and

Banker, 2006).