N-acetyl-L-cysteine ameliorates Autoimmune Encephalomyelitis

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N-acetyl-L-cysteine Ameliorates Autoimmune Encephalomyelitis

in Lewis rats

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Aug 29, 2005 07:56 PDT

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N-acetyl-L-cysteine ameliorates the inflammatory disease process in

experimental autoimmune encephalomyelitis in Lewis rats

http://www.jautoimdis.com/content/2/1/4

 

Romesh Stanislaus1 , Anne G Gilg2 , Avtar K Singh2 and Inderjit Singh2

1Department of Biostatistics, Bioinformatics & Epidemiology, Medical

University of South Carolina, Charleston, SC, USA

2Department of Pediatrics, Medical University of South Carolina,

Charleston, SC, USA

 

Journal of Autoimmune Diseases 2005, 2:4

doi:10.1186/1740-2557-2-4

 

The electronic version of this article is the complete one and can be

found online at: http://www.jautoimdis.com/content/2/1/4

 

Received 1 April 2005

Accepted 3 May 2005

Published 3 May 2005

 

© 2005 Stanislaus et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the

Creative Commons Attribution License

(http://creativecommons.org/licenses/by/2.0), which permits unrestricted

use, distribution, and reproduction in any medium, provided the original

work is properly cited.

--

 

Keywords: EAE, Macrophages, infiltration N-acetyl-L-cysteine, CNS

 

Outline Abstract

 

Abstract

1. Introduction

2. Materials and methods

3. Results

4. Discussion

Acknowledgements

References

 

 

We report that N-acetyl-L-cysteine (NAC) treatment blocked induction of

TNF- & #945;, IL-1 & #946;, IFN- & #947; and iNOS in the CNS and attenuated

clinical disease in the myelin basic protein induced model of

experimental allergic encephalomyelitis (EAE) in Lewis rats.

Infiltration of mononuclear cells into the CNS and induction of

inflammatory cytokines and iNOS in multiple sclerosis (MS) and EAE have

been implicated in subsequent disease progression and pathogenesis. To

understand the mechanism of efficacy of NAC against EAE, we examined its

effect on the production of cytokines and the infiltration of

inflammatory cells into the CNS. NAC treatment attenuated the

transmigration of mononuclear cells thereby lessening the

neuroinflammatory disease. Splenocytes from NAC-treated EAE animals

showed reduced IFN- & #947; production, a Th1 cytokine and increased IL-10

production, an anti-inflammatory cytokine. Further, splenocytes from

NAC-treated EAE animals also showed decreased nitrite production when

stimulated in vitro by LPS. These observations indicate that NAC

treatment may be of therapeutic value in MS against the inflammatory

disease process associated with the infiltration of activated

mononuclear cells into the CNS.

 

 

Outline 1. Introduction

 

Abstract

1. Introduction

2. Materials and methods

3. Results

4. Discussion

Acknowledgements

References

 

 

Multiple sclerosis (MS) is a chronic demyelinating disease marked by

focal destruction of myelin, resulting in the loss of oligodendrocytes

and axons accompanied by an inflammatory disease process [1-3].

Experimental autoimmune encephalomyelitis (EAE) is an animal model of

MS. Both MS and EAE are initiated by a T-cell mediated autoimmune

response (CD4+ and CD8+) against myelin components followed by induction

of inflammatory mediators (chemokines and cytokines) that in turn define

the pattern of perivascular migration of activated T-cells and

mononuclear cells into the CNS [1-4].

 

The sequence of events associated with loss of oligodendrocytes and

myelin in MS and EAE are not precisely understood. A complex interaction

between the mediators released by infiltrating cells and brain

endogenous activated glial cells (astrocytes and microglia) are believed

to contribute towards the inflammatory disease process and tissue damage

[1-3,5-7]. Numerous studies have documented the expression of

proinflammatory cytokines (TNF- & #945;, IL-1 & #946;, and IFN- & #947;) in

EAE and MS tissue and increased levels of IFN- & #947; and TNF- & #945;

levels in CNS or plasma appear to predict relapse in MS [1-3,8]. On the

other hand, enhanced expression of anti-inflammatory cytokines (IL-4,

IL-10 and TGF- & #946;) appears to mediate disease remission [1-3,9]. In

MS brain, expression of iNOS by activated astrocytes, microglia and

macrophages is associated with the demyelinating regions [10-13]. The NO

derived from iNOS as ONOO- (a reaction product of NO and O2-) is thought

to play a role in the pathobiology of MS and EAE. Peroxynitrite (ONOO-)

is able to modify proteins, lipids and DNA resulting in damage to

oligodendrocytes and myelin [1-3].

 

In spite of extensive research to develop pharmacotherapeutic agents to

ameliorate or reduce the number of exacerbations and subsequent

progression of neurological disability in MS, only a few therapies are

available. Presently, IFN- & #946; [14] and glatiramer acetate [15] are

used in treatment of MS but the therapeutic efficacy of these compounds

is limited by significant side effects. Recent studies from our

laboratory [16,17] and others [18] report the potential of HMG-CoA

reductase inhibitors (statins) in attenuating the disease process in

EAE. The efficacy derives from a shift from an inflammatory Th1 response

towards an anti-inflammatory Th2-biased response [16,18,19], blocked

infiltration of mononuclear cells into CNS [20] and attenuation of the

induction of proinflammatory cytokines (TNF- & #945;, IFN- & #947;) and iNOS

in the CNS of EAE animals [17,20].

 

Reactive oxygen species (ROS) and reactive nitrogen species (RNS),

generated as a result of the inflammatory process, are believed to play

a role in the pathobiology of EAE and MS [10,12,13]. Cell culture

studies showed that NAC, a potent antioxidant, inhibited induction of

TNF- & #945; and iNOS and NO production in peritoneal macrophages, C6

glial cells and primary astrocytes, and blocked the activation of

NF & #954;B in peritoneal macrophages [21]. Accordingly, oral

administration of the oxidant scavenger NAC was found to attenuate EAE

clinical disease [22]. The present studies were designed to elucidate

the mechanism of observed therapeutic efficacy of NAC against EAE. These

studies document that NAC treatment inhibited the clinical disease by

attenuating multiple events in EAE disease such as shifting the immune

response from a Th1 bias, increasing IL-10 cytokine production by

splenocytes, attenuating transmigration of mononuclear cells, and

inhibiting induction of proinflammatory cytokines (TNF- & #945;,

IL-1 & #946;, IFN- & #947;) and iNOS in the CNS. Taken together these

results suggest NAC may be of therapeutic value for cell-mediated

autoimmune diseases such as multiple sclerosis.

 

 

Outline 2. Materials and methods

 

Abstract

1. Introduction

2. Materials and methods

3. Results

4. Discussion

Acknowledgements

References

 

 

Chemicals

 

Myelin basic protein (MBP) isolated from guinea pig brain and complete

Freund's adjuvant (CFA) and pertussis toxin were obtained from Sigma

(St. Louis, MO). N-acetyl-L-cysteine (NAC) was obtained from Calbiochem

(USA).

 

EAE induction and treatment with NAC in Lewis rats

 

Experiments were performed on female Lewis rats (Harlan Laboratory, USA)

weighing 250–300 g. Animals were housed in the animal care facility of

the Medical University of South Carolina, USA, throughout the experiment

and provided with food and water ad libitum. All experimental protocols

were reviewed and approved by the Institutional Animal Care and Use

Committee. EAE was induced by subcutaneous injection of 50 & #956;g of

MBP (per animal) emulsified in complete Freund's adjuvant in the region

of the footpad of the hind leg on day 1 followed by a booster injection

of the same on day 7. Additionally, animals received 200 ng of pertussis

toxin on days 0 and 1. Clinical signs in these rats manifest as

ascending paralysis resulting in EAE in most animals. The clinical signs

of EAE were scored by a masked investigator as 0 = normal; 1 =

piloerection; 2 = loss in tail tonicity; 3 = hind leg paralysis; 4 =

paraplegia; and 5 = moribund. NAC treatment was started on the first day

of immunization (day 1) and continued daily for the duration of the

experiment. One group of rats induced for EAE (n = 15) was given

intraperitoneal injections of NAC (150 mg/kg body weight in PBS with pH

adjusted to 7.2 with NaOH). The second group of rats (n = 15) was

induced for EAE and treated with the vehicle (PBS). Animals receiving

only CFA were used as the control group (n = 15). Untreated EAE animals

were sacrificed at clinical stage 4 (paraplegia) or 5 (moribund)

according to approved protocol. NAC treated animal group was sacrificed

at their peak clinical disease, which was an average clinical score of

3, as determined from preliminary studies. Tissue for histology and

immunohistochemistry and splenocytes were recovered for analysis.

 

Histopathology-Immunohistochemistry

 

The lumbar region of the spinal cord was dissected and carefully

processed for histological and immunohistological examination (n = 12).

Spinal cords were fixed in 10% buffered formalin (Stephens Scientific,

Riverdale, NJ), embedded in paraffin and sectioned at 4 & #956;m

thickness. Sections were then stained for various cytokines and cell

markers.

 

Immunohistochemistry for TNF- & #945;, IFN- & #947;, IL-1 & #946;, iNOS and

nitrotyrosine was done as previously described [17]. Sections were

incubated with appropriate antibodies (1:100) overnight followed by

fluorochrome conjugated secondary IgG antibody (1:100, Sigma, St. Louis,

MO) and mounted with Fluoromount G (EMS, Fort Washington, PA).

Non-immune IgG was used as control primary antibody. Sections were also

incubated with TRITC or FITC conjugated IgG without the primary antibody

as negative control. Nuclear staining was performed using DAPI (Sigma,

St. Louis, MO) and hematoxylin and eosin (H & E) staining was performed as

described by Kiernan, J.A (1990). All the sections were analyzed using

an Olympus microscope (Olympus BX60, Opelco, Dulles, VA) and images were

captured using a digital video camera (Olympus U-CMAD-2, Optronics,

Galeta, CA) and Adobe Photoshop (Adobe Systems, CA).

 

Quantitative analysis of infiltrating cells

 

Infiltrating cells labeled with either ED1 or DAPI were analyzed using

Image-Pro Plus 4.0 (Media Cybernetics, Maryland, USA) software.

Individual sections were analyzed and the mean and SD were calculated

for each group (n = 12). The group means were compared and the

significance of difference was determined. A p value of <0.05 was

considered significant. This analysis was done using the Regression Data

Analysis tool of Microsoft Excel 4.0 (Microsoft, Redmount, WA).

 

Splenocyte Isolation and Cell Culture

 

Splenocytes were isolated from each animal group (Control, EAE, EAE+NAC)

(n = 6) using Lympholyte®-Rat (Cedarlane Laboratories Ltd., Hornby,

Canada) density separation medium according to manufacturer's

instruction. The cell concentration in the suspension was adjusted to 2

× 107 nucleated cells per ml or less, layered on Lympholyte®-Rat density

separation medium, and centrifuged for 20 min at 1000 g – 1500 g at room

temperature. The interface formed after the centrifugation was then

extracted using a Pasteur pipette, and transferred to a new centrifuge

tube. The transferred cells were then diluted with medium, and

centrifuged at 800 g for 10 min, washed twice with media, and cultured

in 24-well plates at a concentration of 5 × 106 cells/ml. The cells were

then stimulated in vitro with MBP (20 & #956;g/mL), LPS (1 & #956;g/mL),

or PHA (10 & #956;g/mL), (Sigma, St. Louis, MO, USA), or without any

stimulants for 48 hrs. Each treatment was performed in triplicate. At

the end of the 48 hr. incubation period, supernatants were collected and

used for the measurement of cytokines and nitrite.

 

ELISA

 

Cytokines (IFN- & #947; and IL-10) were detected in culture supernatants

using commercially available OptEIA™ kits from PharMingen (San Diego,

CA, USA) according to manufacturer's instructions. The assay procedure

is as follows: 96-well microplates were coated with capture antibody

diluted in coating buffer overnight at 4°C. Plates were then washed and

blocked with assay diluent (PharMingen, San Diego, CA, USA) for 1 hr at

room temperature. Blocked plates were then washed, and the standards and

samples added to the wells and incubated for 2 hr. at room temperature.

At the end of incubation, plates were washed and working detector

(detection antibody + Avidin-HRP) was added to the wells and incubated

for 1 hr. at room temperature. Following incubation, plates were washed

and TMB substrate reagent was added (PharMingen, San Diego, CA, USA) to

the wells for 30 min. at room temperature in the dark. At the end of the

incubation, stop solution (1 M H3PO4) was added, and absorbance read at

450 nm using a Spectramax® microplate spectrophotometer (Molecular

Devices, Sunnyvale, CA, USA).

 

Nitrite measurement

 

Nitrite levels were determined on isolated splenocytes with Griess

reagent as previously described [23] with minor modifications. One

hundred & #956;l of culture supernatant was allowed to react with 100

& #956;l of Griess reagent and incubated at room temperature for 15 min.

The optical density of the assay samples was measured at 570 nm using a

96-well plate Spectramax® microplate reader with SOFTMAX® software

(Molecular Devices, Sunnyvale, CA, USA). Fresh culture media served as

the blank in all experiments. Nitrite concentrations were calculated

from a standard curve derived from the reaction of NaNO2 in the assay.

 

 

Outline 3. Results

 

Abstract

1. Introduction

2. Materials and methods

3. Results

4. Discussion

Acknowledgements

References

 

Figures

 

Figure 1

The protective effect of NAC on the clinical signs of MBP induced EAE in

female Lewis rats

 

 

 

Figure 2

Inflammation and demyelination in sections of lumbar spinal cord from

control, EAE and EAE + NAC (n = 12) treated Lewis rats

 

 

 

Figure 3

Quantification of the inflammatory infiltrates by immunostaining of

Lewis rat spinal cord (n = 12)

 

 

 

Figure 4

Immunofluorescent detection of IFN- & #947;, TNF- & #945;, IL-1 & #946;, iNOS

and nitrotyrosine in the CNS of female Lewis rats

 

 

 

Figure 5

IFN- & #947;, IL-10 and nitrite production by splenocytes from control,

EAE and treated animals

 

 

 

 

Effect of NAC on the Clinical Signs of Rats

 

Our goal was to investigate the effect of NAC on rats induced for acute

EAE. In the Lewis rat model MBP induces an acute monophasic disease

progression. As shown in Fig. 1, clinical signs of EAE were evident in

MBP-treated Lewis female rats from the 8th day after first immunization

inducing an acute monophasic disease progression resulting in paraplegia

(clinical score of 4) or moribund state (clinical score of 5) on or

around the 12th day. However, the control animals receiving only

complete Freund's adjuvant did not show any disease symptoms (Fig. 1).

Animals induced for EAE but given only the vehicle closely followed the

disease progression of MBP-treated rats. Treatment of MBP-injected rats

with NAC, administered from the first day of immunization, protected the

rats by attenuating the severity of disease progression (Fig. 1). NAC

treated animals had milder clinical signs (average clinical score of 3

as compared to 5 for EAE).

 

Effect of NAC on the infiltration of inflammatory cells into the spinal

cord

 

The neuropathological changes in EAE and MS are associated with the

blood brain barrier breakdown and infiltration by mononuclear cells

[24,25]. Clinical disease in EAE has been shown to correlate with the

invasion of CNS by mononuclear cells. These studies demonstrate that

MBP-induced EAE results in the induction of inflammatory disease, and

treatment with NAC provides protection against the EAE disease process.

Therefore, in order to understand the mechanism of therapeutic efficacy

in EAE, we studied the effect of NAC on the invasion of mononuclear

cells into the CNS in the EAE model.

 

The spinal cords of rats induced for EAE had heavy mononuclear

inflammatory infiltrates on the meningeal surfaces, perivascular areas

and interstitial areas as seen by H & E staining (Fig. 2a). EAE animals

treated with NAC showed infiltration of the CNS by inflammatory cells

but not to the extent as that seen in EAE animals. Further analysis of

the cell infiltrates was performed to identify the major cell type

infiltrating the CNS in addition to the T-cells. Immunohistochemical

methods using ED1 (monocyte/ macrophage marker) and DAPI (for nucleated

cells) were performed. As seen in Fig. 3, EAE animals showed the most

infiltration by ED1 positive cells. In contrast the NAC-treated animals

showed significantly less infiltration by ED1 positive cells (reduced by

an average of 46 percent). Quantitative analysis of cell infiltrates

into the CNS showed a significant amount of nucleated as well as ED1

positive cells in the CNS of EAE animals (Fig. 3b). In contrast, cell

infiltration into the CNS of treated animals was significantly less than

that seen in EAE animals (reduced by an average of 45 percent).

 

Effect of NAC on the expression of pro-inflammatory cytokines and iNOS

in the spinal cord

 

Since the major source of IL-1 & #946; in EAE is monocytes/macrophages, as

further evidence for macrophage infiltration we examined the expression

of IL-1 & #946; in the CNS. As evidenced by Fig. 4c, expression of

IL-1 & #946; was evident in the CNS of EAE induced animals and to a far

lesser degree in the NAC treated animals. IL-1 & #946; expression was also

co-localized to ED1 positive cells in EAE animal spinal cords (data not

shown). We also examined the expression of proinflammatory cytokines

(TNF- & #945; and IFN- & #947;), iNOS and nitrotyrosine in the spinal cord

sections from control, EAE, and NAC-treated EAE rats using

immunohistochemistry. As seen in figure 4a–e, MBP-induced EAE resulted

in the expression of TNF- & #945;, IFN- & #947;, IL-1 & #946;, IFN- & #947;,

iNOS and nitrotyrosine. NAC treatment of EAE blocked the induction of

these cytokines, iNOS, and nitrotyrosine similar to control animals.

 

IFN- & #947;, IL-10 and nitrite production by splenocytes from EAE and

treated animals

 

In vitro splenocytes assays were performed to elucidate whether NAC

treatment could cause a shift to Th2-type T-cell activity. In order to

examine this effect, we studied the effect of NAC on the major Th2

cytokine in the EAE disease process, IL-10. Splenocytes (8 × 105 cells

per well) were obtained from Control, EAE, and EAE + NAC treated rats.

Cells were stimulated in vitro with PHA (10 & #956;g/ml, a & b), MBP (20

& #956;g/ml, a & b) or LPS (1 & #956;g/ml, c) for 48 hrs. The levels

(pg/ml) of IFN- & #947; and IL-10 in culture supernatants were measured

using ELISA kits. As seen in Fig. 5 there was a significant increase in

IFN- & #947; (5a) and decrease in IL-10 (5b) in splenocytes from untreated

EAE animals. NAC treatment reduced IFN- & #947; production by splenocytes

(by 59% for PHA and 40 % for MBP) and up-regulated IL-10 production by

EAE splenocytes (by 31% for PHA and 34% for MBP). Culture supernatants

were collected and accumulated nitrite, a stable product of NO

production, was measured using Griess reagent. NAC treatment also

inhibited the production of nitrite by LPS-stimulated splenocytes by 71%

as compared to splenocytes from EAE animals. These studies indicate that

NAC treatment reduced IFN- & #947;, a proinflammatory Th1 cytokine and

increased IL-10, an anti-inflammatory cytokine.

 

 

Outline 4. Discussion

 

Abstract

1. Introduction

2. Materials and methods

3. Results

4. Discussion

Acknowledgements

References

 

 

The evidence presented in this paper demonstrates that NAC treatment

reduced the inflammatory monocyte/macrophage cells in the CNS of Lewis

rats with acute monophasic EAE. This in turn results in protection both

in terms of clinical and histopathological changes. These conclusions

are based on the following observations. 1) NAC treatment of EAE rats

reduced the severity of EAE clinical symptoms, 2) attenuated the

infiltration of mononuclear cells into the CNS of EAE rats, 3) blocked

the induction of proinflammatory cytokines, iNOS and nitrotyrosine in

the CNS, and 4) decreased proinflammatory Th1 cytokine responses

(IFN- & #947;) from ex vivo splenocytes while increasing anti-inflammatory

cytokine production (IL-10), and decreasing NO production in

LPS-stimulated splenocytes.

 

The infiltration of activated mononuclear cells into the CNS of EAE is a

critical event in the progression of the disease [26]. We have shown

both qualitatively and quantitatively that ED1 positive leukocytes,

namely macrophage/monocytes, were significantly decreased in animals

treated with NAC as compared to the EAE animals. This decrease also

correlated with the amelioration of clinical disease in female Lewis

rats. As compared to our previous studies with lovastatin, NAC was not

as effective in blocking the transmigration of inflammatory cells (NAC

reduced by an average of 46%, while lovastatin reduced by 85%) and hence

did not delay the onset of disease as was achieved with lovastatin

treatment (EAE, EAE + NAC onset day 8 versus EAE + lovastatin onset day

11). However, NAC reduced the clinical scores to the same levels as

those obtained with lovastatin (both had clinical scores maximum of 3).

Other studies have also shown a correlation between macrophage

infiltration and EAE clinical disease [27]. Inflammatory cytokine

expression (IFN- & #947;, IL-1 & #946;, and TNF- & #945;) was also inhibited

in the CNS of EAE animals treated with NAC. As a consequence, inhibition

of IFN- & #947; expression in NAC treated animals could in turn result in

the reduced expression of MHC II molecules thereby inhibiting the

proliferation of T-lymphocytes as has been shown with statins [28,29],

copolymer 1 [30] and IFN- & #946; [31].

 

Evidence indicates that iNOS while not a crucial factor for induction of

EAE, plays a major role in the progression of the disease. The critical

factors is the amount of NO produced that tips the balance in favor or

against the pathogenesis of EAE [32]. The peroxynitrite (ONOO-) produced

by reaction of NO and O2- can damage membranes and cells by

nitrosylation of lipids, proteins and nucleic acids. The induction of

IL-1 & #946; and activation of NF & #954;B were shown to precede the

induction of iNOS in ED1+ cells [33]. Here we report that NAC blocked

the induction of IL-1 & #946; in the CNS of EAE animals. Ex vivo studies

using splenocytes isolated from control, EAE and EAE+NAC treated animals

showed that NAC inhibited IFN- & #947; production while increasing IL-10

production. These changes coincided with a decreased NO production in

the cultured splenocytes. NAC treatment was not as effective as

lovastatin in altering cytokine production, but the reduction in nitrite

was identical. NAC treatment reduced IFN- & #947; production by

splenocytes (NAC by 59% and 40%, LOV by 76% and 60% for PHA and MBP

respectively) and up-regulated IL-10 production by EAE splenocytes (NAC

by 31% and 34%, LOV by 350% and 490% for PHA and MBP respectively). NAC

treatment also inhibited the production of nitrite by LPS-stimulated

splenocytes by 71% as compared to splenocytes from EAE animals. These

studies indicate that NAC treatment inhibited a proinflammatory Th1

biased cytokine response (IFN- & #947;) while promoting an increase in

IL-10, an anti-inflammatory cytokine. Similar shifts from Th1 cytokine

profile to Th2 have been correlated with disease recovery or improvement

in both EAE and MS [16,18,19,34-37].

 

The brain is particularly vulnerable to oxidative stress due to its high

consumption of oxygen and glucose, enrichment in unsaturated fatty acids

that are subject to oxidation, and presence of regions enriched in iron

and ascorbate that are potent pro-oxidants for brain membranes.

Moreover, higher levels of glucose upregulate the neuroinflammatory

process measured as induction of iNOS and NO production [38]. Coupled

with the relatively reduced antioxidant defenses in the brain, exposure

of brain cells to reactive oxygen or nitrogen species can be detrimental

and is thought to contribute to the pathogenesis of many brain disorders

[39]. Oxidative stress is important in the etiology of EAE and is

thought to contribute directly to CNS damage [7,40]. N-acetyl-L-cysteine

(NAC) as cysteine, a precursor of glutathione, is a potent anti-oxidant.

By scavenging superoxide radicals, metallothionein and other

antioxidants such as cysteine, N-acetyl-cysteine and glutathione offer

neuroprotection [41]. In vivo NAC enhances hippocampal neuronal survival

after transient forebrain ischemia in rats [42]. Partial protection of

neurons from the dopaminergic neurotoxin

N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was achieved by four

different antioxidants including NAC in the mouse [43]. NAC also has a

protective effect in pneumococcal meningitis in the rat [44]. In vitro,

NAC promotes oligodendrocyte survival in the presence of toxic stimuli

or due to withdrawal of growth factors [45] and maturation of

oligodendrocytes [46]. NAC inhibits Theiler's virus-induced NO and

TNF- & #945; production by murine SJL/J astrocyte cultures [47]. NAC

treatment prevented cytokine-induced decrease in GSH and degradation of

sphingomyelin to ceramide, also blocked cytokine-mediated ceramide

production in rat primary oligodendrocytes, microglia, and C6 glial

cells, thereby preventing cell death. These results suggest that

intracellular levels of GSH may play a critical role in the regulation

of cytokine-induced generation of ceramide leading to apoptosis of brain

cells in demyelinating diseases. [48]

 

In summary, the ability of NAC to inhibit the induction of

proinflammatory cytokines and inhibit the transmigration of inflammatory

cells into the CNS of EAE-induced rats identifies it as a potential drug

for the treatment of neuroinflammatory diseases and possibly other

Th1-mediated autoimmune diseases. In addition, in vitro studies suggest

that NAC may also promote survival of neurons and oligodendrocytes and

thereby potentially facilitating remyelination. MS is a multifactorial

disease and the etiology of the disease in unknown. Consequently, the

targets for the prevention of the disease are currently unknown. However

the disease signs and causes of these are known. For example an increase

in pro-inflammatory cytokines and iNOS activity has been linked increase

in clinical sign. As evidenced in the manuscript, NAC can inhibit the

production of inflammatory cytokines and nitrotyrosine in the CNS during

EAE pathogenesis. Thus, NAC holds out to be a promising therapeutic

agent for the amelioration of MS/EAE.

 

 

Acknowledgements

 

 

 

We would like to thank Joyce Bryan and Carrie Barnes for laboratory

assistance, and Hope Terry for secretarial assistance. This work was

supported by the grants (NS-22576, NS-34741, NS-37766, and NS-40810)

from National Institutes of Health.

 

 

Outline References

 

Abstract

1. Introduction

2. Materials and methods

3. Results

4. Discussion

Acknowledgements

References

 

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