Development of an in Vitro Model for Activation of Satellite Glial Cells in The

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Development of an in vitro Model for Activation

of Satellite Glial Cells in the Sympathetic

Nervous System

Master’s Thesis

Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Graduate Program in Molecular and Cell Biology Susan Birren, Advisor

In Partial Fulfillment Of the Requirements for the Degree Master of Science in Molecular and Cell Biology

by Surbhi Sona February 2014

Acknowledgement

I would like to thank my advisor Dr. Susan Birren for giving me this learning opportunity in her lab. I thank her for her constant guidance and support and for critiquing my work. I also thank her for her insightful questions and suggestions.

I am extremely grateful to Dr. Joana Enes for mentoring my work and training me in the various techniques used in this research. I thank her for her patience and valuable guidance which made this a wonderful learning experience for me. I deeply thank her for providing me with some of the materials used in my experiments - She established the primary rat cultures used in this study. The various HEK cell constructs, used in the neurotrophin experiments, were also developed by her.

I also thank all the members of the Birren lab for their support and valuable discussions on my work.

Abstract

Development of an in vitro Model for Activation of Satellite Glial Cells in the

Sympathetic Nervous System

A thesis presented to the Graduate Program in Molecular and Cell Biology

Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Surbhi Sona

Glial cells of the central nervous system are activated upon injury and modulate the course of several diseases. The glia population of peripheral nervous system is much less explored in this area. I used satellite glial cells (SGCs) from superior cervical ganglia of P0 to P2 rat pups, to develop an in vitro model to study glia activation in the sympathetic nervous system. Several factors including ATP, endothelin-1, lipopolysaccharides and fractalkine have been reported to activate other glia populations and were tested on SGC cultures. Out of these, LPS showed a relatively better consistency in activating SGCs and was used for most of the experiments. To assess activation of SGCs, the cell lysates were analyzed by western blot to check for GFAP expression, a common glia activation marker. There was only a slight difference in GFAP levels between control and treated cells.

Immunocytochemistry was used as an alternative approach to assess SGC activation, comparing cell proliferation between the control and activating conditions. The results suggested cell proliferation to be a better assay than GFAP expression to study SGC activation in cell culture. It is also interesting to look for secretary factors released by SGCs

iii

and to observe if this expression profile alters in response to activation. Using western blot,

I validated production of neurotrophins such as NGF and BDNF in the SGC culture. The expression of these factors altered non-significantly in response to activation. The young and adult animals may have different protein expression profile. So, more SGC-specific activation protocol/modulator; SGC culture from adult animals; and a better activation marker for SGC might be required to develop a robust in vitro model to study significance and implications of activation of SGCs in sympathetic nervous system function. These inferences would be valuable as the sympathetic neurons innervate several vital organs such as heart and activation of glia may contribute in affecting the sympathetic drive to these organs.

Table of Contents

1. Abstract iii 2. List of Tables vi 3. List of Figures vii 4. List of Abbreviations viii 5. Introduction 1 6. Materials and Methods 5 7. Results 11 8. Discussion 22 9. Bibliography 27

List of Tables

Table 1 Activation factors used on satellite glial cells 7

Table 2 Determining the effective concentration and the treatment 7 duration of the activators for satellite glial cells

List of Figures

Figure 1 Design of the Experiment 6

Figure 2 NGF and BDNF expression in satellite glial cells of superior cervical ganglia 12 dissected from P0 to P2 rats

Figure 3 GFAP expression in SGCs in response to the activators – ATP, ET-1 and LPS 14

Figure 4.1 Satellite glial cell proliferation in response to LPS 16

Figure 4.2 Quantification of SGC cell proliferation in response to LPS 17

Figure 5 Expression of NGF and BDNF in SGCs in response to LPS treatment 19

Figure 6 TNF-α levels in SGC culture media in response to short term treatment 21 with LPS and FRK

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List of Abbreviations

ATP Adenosine Tri-phosphate

BDNF Brain Derived Neurotrophic Factor

CNS Central Nervous System

DAPI 4’,6-diamidino-2-phenylindole

DIV Day(s) in vitro

ET-1 Endothelin-1

FRK Fractalkine

GFAP Glial Fibrillary Acidic Protein

LPS Lipopolysaccharides

NGF Nerve Growth Factor

PNS Peripheral Nervous System

SCG Superior Cervical Ganglia

SGCs Satellite Glial Cells

SMEM Minimum Essential Media for Suspension culture

TNF-α Tumour Necrosis Factor – α

viii

Introduction

First described by Rudolf Virchow in 1856, glia were thought to be accessory cell population that simply supported the neurons [Freeman et al., 2013]. Much later, glial cells received the much deserved attention, when through many independent researches studies, it was proposed and established that glia are both affected by as well as contribute significantly to various neuropathological conditions such as Multiple Sclerosis, Epilepsy, Alzheimer’s

Disease and Schizophrenia, Parkinson’s Disease, Huntington’s Disease and Amyotrophic

Lateral Sclerosis [Miller, 2005 and Lobseiger and Cleveland, 2007].

The glia population in the central nervous system (CNS) comprises of astrocytes, oligodendrocytes and microglia; while Schwann cells and satellite glial cells (SGCs) are the major glia population in the peripheral nervous system (PNS). Relatively much less is described about glia population in the peripheral nervous system as the initial glia research was focussed on glia population of the central nervous system [Ji et al., 2013].

Sympathetic ganglia of PNS relay inputs from CNS to several visceral organs. These ganglionic structures consist of sympathetic neurons enveloped by satellite glial cells

[Hanani, 2010]. SGCs share several similarities with astrocytes such as expression of the same glial markers (GFAP and S100) and formation of gap junctions [Ji et al., 2013]. Thus,

astrocyte research may provide useful cues for developing a better understanding of the functional significance of SGCs and their role in human pathophysiology. In this context, it is important to mention that sympathetic dysfunction has been implicated in several diseases such as diabetes, hypertension and chronic heart failure.

Brain injury activates astrocytes which is manisfested as increased expression of glial fibrillary acidic protein (GFAP) [McMillan et al., 1994]. SGCs in sensory ganglia [Zhang et al.,

2009 and Takeda et al., 2009] and in sympathetic ganglia [Elfvin et al.,1987 and Zigmond et al., 2007] also become reactive post injury or inflammation in vivo.

Activated astrocytes express elevated levels of a wide range of molecules – structural proteins such as GFAP; growth factors such as NGF; cytokines such as interleukins and TNF-

α; and enzymes such as glutamine synthetase, to name a few [Ridet et al., 1997]. Thus, upregulation of one or more of these molecules serve as activation marker for astrocytes and given the similarities between astrocytes and SGCs, some of these may also serve as activation markers for SGCs of sympathetic system. The glia cells also undergo increased proliferation in response to activation [Jasmin et al.,2010].

LPS, a constituent of cell wall of Gram negative bacteria, is widely used to induce activation in astrocytes and microglia, both in vitro as well as in vivo [Zhong et al., 2012 and Yao et al.,

2014]. Similarly, astrocytes have been described to turn reactive in response to purinergic signalling mediated by ATP [Tran and Neary, 2006]. ET-1 has been previously described in

relation to reactive gliosis in CNS. It is a vasoconstrictor produced by astrocytes in response to injury and has autocrine effect. A study has previously demonstrated that ET-1 promotes activation of astrocytes [Gadea et al., 2008]. Activation of PNS glia is not as extensively studied. A recent work reported activation of SGCs in sensory ganglia using Fractalkine, which is a chemokine [Souza et al., 2013].

Neurotrophins are target derived factors that promote growth and survival of neurons in the peripheral nervous system. They are also known to be modulators of synaptic properties

[Poo 2001]. Under pathological conditions, the expression of these factors is altered, for example, in the heart [Govoni et al., 2011]. Apart from the peripheral organs, SGCs of sensory ganglia also produce neurotrophins [Gosselin et al., 2010]. Since glia get activated by injury and other pathological conditions, it would be interesting to explore if that activation has implications on neurotrophin expression by SGCs which in turn would modulate synaptic properties.

Since Sympathetic neurons innervate vital organs and such a study could provide useful insights on role of glia in disease pathogenesis and progression. An in vitro model for SGC activation in sympathetic system could help identify factors that mediate such activation.

Because in vitro models eliminate complexities of an in vivo process, the SGC culture model could help characterize response of SGC to activation. The mechanisms involved can also be characterized in such models. This would further aid in understanding satellite glial cell

biology and the implications of their activation in response to injury, inflammation and other conditions that activate glial cells.

Materials and Methods

Cell Culture

Superior cervical ganglia were dissected from postnatal rats (P0 to P2). The ganglia were incubated 1 hour at 370C in SMEM (GIBCO) supplemented with 350U/mL of collagenase type I (CLS-1, Worthington) and 7.5 U/mL of dipase (Gibco). The digested ganglia were then transferred to 15 mL tube containing filter sterilized MAH food (25 mL Fetal Bovine Serum;

10 mL 1:1:2 (0.3% Dextrose, 0.5% glutamine and 0.5% Penn/Strep (GIBCO) in water); and 5 mL FVM (10 µg/mL DMPH4 (Calbiochem), 50 µg/mL glutathione and 1 mg/mL ascorbic acid in distilled water; set pH between 5-6 using 0.5 N KOH) in 250 mL L15CO2) and centrifuged for 5 minutes at 210 g. The cell pellets were resuspended in 5 mL MAH food using fire polished Pasteur pipettes and plated in a 60 mm plastic preplating dish (Bolite 35mm,

Thermo Scientific). The primary culture was incubated at 370C for 1-1.5 hours. The cells and the media were pipette out into a 15 mL tube and centrifuged for 7 minutes at 210 g. Again, the cell pellets were resuspended in 5 mL MAH food as described and plated at a density of

75,000 cells per dish in 10 mm culture dishes (Corning Incorporated) containing 10 mL of

MAH food supplemented with 5µg/mL of NGF (BD Biosciences). The dishes were coated with collagen (BD Biosciences) and laminin (BD Biosciences) before plating. Half of the medium was replaced with fresh MAH food (supplemented with 5µg/mL NGF) every alternate day. These procedures yielded co-cultures of neurons and satellite glial cells.

To derive relatively pure glial culture, 7-8 DIV (days in vitro) cultures were trypsinized and plated in dishes coated with collagen and laminin (60mm dishes (Thermo Scientific) for

Western Blotting; 35 mm glass bottom dishes (MatTek) for Immunostaining). MAH food without NGF was used for culturing and the cells were plated at a density of 200,000 cells per dish for 60 mm dishes and 45,000 cells per dish for 35mm dishes, in NGF free MAH food.

Half of the media was changed every alternate day till cells reached optimal density for treatment (semi-confluent to confluent depending on duration of the treatment). In 2-3 days, most neurons died off in absence of NGF.

Figure 1. Experimental Design (a) Glia in co-culture with neurons grown for 7 day in vitro (without trypsinization). Half the media was changed every alternate day till they were lysed for western blot or fixed for immunostaining. (b) Pure glial culture (trypsinized at 7div)grown for additional X days. ATP (100µM), ET-1 (200nM) or LPS (1µg/mL) were added to the respective culture dishes when the cells were somewhat confluent (at 3DIV). The modulators were added every alternate day for a duration of 4 days. (c) The experiment design was the same as described in (b) except the treatment duration. Here, the SGCs were subjected to different concentrations of the modulators (Table 2) for a period of 6 to 24 hours.

Treatment

Common modulators, that have been used to activate glia population, were used to activate

SGCs at the following concentrations:

Table 1. Activation Factors used on Satellite Glial Cells

S. No. Activator Concentration Reference 1. ATP 100 µM Gandelman et al., 2010 2. ET-1 200 nM Gadea et al., 2008 3. LPS 1 µg/mL Lin et al., 2008 4. FRK 100 ng/mL Souza et al., 2013

Table 2. Determining effective concentration and duration of activator for satellite glial cell treatment

S. No. Activator Concentration Duration

6 h 0.5 µg/mL 12 h 24 h 6 h 1. LPS 1 µg/mL 12 h 24 h 6 h 2 µg/mL 12 h 24 h 6 h 2. FRK 100 ng/mL 12 h 24 h

Immunocytochemistry

For the purpose of immunostaining, trypsinized cultures were replated in glass-bottomed culture dishes (MatTek 35 mm) at a density of 45,000 cells per dish. When the cells were semi-confluent ( 5-6 days), they were fixed using 4% paraformaldehyde (PFA) for 15 minutes. They were then permeabilized by incubating them in permeabilizing solution (0.1%

NP40 or Triton X and 1% Donkey Serum (Gibco) in PBS) for 10 minutes. This was followed by

1 hour of blocking using 10% Donkey Serum and overnight incubation in primary antibody

(rb-Ki67; 1:1500, Abcam) at 40C. Excess antibody was washed off using PBS. Incubation in secondary antibody (dk anti-rb-FITC; 1:5000; Abcam) was carried out at room temperature for 1 hour in dark. The cells were also incubated in DAPI (Invitrogen Life Technologies) for 10 minutes to stain the nuclei. The cells were rinsed twice with PBS and 1 mL of N-propyl gallate (NPG) was added to protect them from photobleaching. The cells were observed under 60X oil immersion objective of Olympus IX81 inverted microscope.

Western Blotting

The cultured cells were rinsed twice with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4 ). About 170 μL of RIPA lysis buffer (50mM

Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, 2mM EDTA and Complete Mini

Protease inhibitor cocktail (Roche Diagnostics, Germany, #1836170001)) was added to each dish to homogenize the cells. After 30 minute incubation in the lysis buffer at 40C, the cell lysate was passed through 22-gauge needle and centrifuged (9300 g ,5 min, 40C) to collect the supernatant. Protein concentrations were determined by Bradford Assay

Samples were subjected to SDS-PAGE and transferred to either Nitrocellulose membrane

(0.2 μM, Biorad) or PVDF membrane (0.2, μM, Biorad). The membranes were blocked using non fat dry milk in phosphate buffered saline (for nitrocellulose 5% milk solution while 10% for PVDF membrane). The membranes were incubated in respective primary antibodies for

2 hours at room temperature: m-GFAP (1:500; Sigma #G3893), rb-NGF (1:500; Santa Cruz

#sc-548), rb- BDNF (1:500; Santa Cruz #sc-546), rb-actin (1:5000 and 1:7500; Odyssey #

92642210). The membranes were then washed 4 times with phosphate buffered saline with

1% Tween 20 (1% PBST) for 5 minutes each and incubated in appropriate horseradish peroxidise conjugated secondary antibody (gt anti-m HRP (1:7500; Jackson

ImmunoResearch #111035144) or gt anti-rb HRP (1:7500; Jackson ImmunoResearch

#111035144)) for 1 hour at room temperature. 1% PBST was used to wash off secondary antibody. Blots were developed using LumiGLO Chemiluminescent Substrate (KPL# 546100) on Blue Devil X-ray Films (Genesee Scientific #30-100). Both primary and secondary antibodies were diluted in 1% PBST.

Band intensity was quantified using Biorad Gel doc XR system. Protein bands were normalized by bands of loading control (actin)

ELISA

Quantikine®ELISA Rat TNF-α Immunoassay kit (RnD System) was used to quantify TNF-α in the culture medium. Two of the sample sets (‘Control’ and ‘FKN 6 hour treatment’) were

concentrated 3 times using Vivaspin 20 columns (5000 MWCO PES membrane; Sartorius

Stedium Biotech, Germany). All samples were diluted 3 times using assay diluents. 50 µL of serially diluted standards (800 pg/mL to 0 pg/mL), diluted samples and TNF-α control were added in duplicates to wells pre-coated with rat TNF-α antibody and incubated at room temperature for 2 hours. This was followed by 5 washes to remove excess antibody. 100 µL of rat TNF-α conjugated to Horse Radish Peroxidase was added to each well and incubated for 2 hours at room temperature. Again 5 washes were performed to remove excess antibody. 100 µL of substrate solution (equal volumes of hydrogen peroxide and tetramethylbenzidine; freshly mixed) was then added to each well and incubated for 30 minutes in dark at room temperature. Reaction was stopped using 100 µL of stop solution

(dilute hydrochloric acid) and readings taken within 30 minutes

Results

1. Satellite glial cells of sympatheic ganglia express neurotrophins:

Neurotrophic factors such as NGF and BDNF have been found to enhance synaptic

transmission of postganglionic sympathetic neurons [Luther et al., 2013]. Satellite glial

cells may be important local source of neurotrophins in the sympathetic ganglia, and

their expression might be altered upon injury. To check whether satellite glial cells

express neurotrophins, cultured glial cells were lysed and NGF and BDNF expression was

assessed using western blot. HEK cells (HEK 293T) were transfected with NGF and BDNF

plasmids for overexpression these proteins. These served as positive controls in the

respective western blots. HEK cells transfected with empty vector was used as negative

control. Pure NGF and BDNF were used as controls for the mature form of these

proteins.

Polyclonal antibodies were used which detected all the premature as well as mature

forms of NGF and BDNF. The SGCs expressed the premature form of NGF and BDNF. All

the HEK cells seem to express both NGF as well as BDNF at some basal level. However,

the HEK cells carrying the neurotrophin plasmid construct exhibit overexpression of the

respective proteins which validated the antibody used and also served as positive

control. The mature form of BDNF was observed in the glia conditioned media derived

from the SGC culture indicating that the SGCs secrete this factor. The mature NGF band was missing in the GCM lane. This could be due to undetectable levels of NGF as the lysate from SGCs also show very low levels of NGF as compared to the BDNF blot.

(Figure 2).

Figure 2. NGF and BDNF expression in satellite glial cells of superior cervical ganglia dissected from P0 to P2 rats. Actin was used as loading control. (a) NGF is expressed by SGCs. Cell lysate from HEK cells transfected with NGF plasmid construct was used as positive control as well as to validate the NGF antibody. Lysate from HEK cells transfected with empty vector and BDNF plasmid contruct were used as negative controls. These cells, however, still show basal level expression of NGF and were not a true negative control. Pure NGF was used as positive control for secretary form of NGF. Glia conditioned media was analyzed to detect NGF secreted by SGCs. The secretary form of NGF in GCM could not be detected. (b) BDNF is expressed by SGCs. HEK cells transfected with empty vector and BDNF plasmid construct were used as negative and positive controls, respectively. Secretary form of BDNF was detected in the glia conditioned media from two independent cultures.

Just like the target tissues of sympathetic system, the SGCs also express the

neurotrophic factors NGF and BDNF. This is indicative of their possible contribution in

modulating the synapse in the sympathetic nervous system.

2. Activation of SGCs of sympathetic ganglia by various modulators

Various modulators, that have been previously described to activate glia population,

were used in order to activate SGCs. Relatively pure SGC culture were established by

trypsinizing 7 day old SGC-neuron co-culture and then replating them without NGF to

eliminate neurons. ATP (100µM), ET-1 (200nM) and LPS (1µg/mL) were added to

respective dishes. The treatment continued for 4 days and the modulators were

replenished every alternate day (Figure 1). The cell lysates were subjected to western

blot for GFAP. Actin was used as loading control (Figure 3 a). The bands were quantified

using BioRad Gel Doc system. GFAP content was normalized over actin. The values were

then expressed as fold change by dividing each of them by control. Data from two

independent experiments was used to plot graph (Figure 3 b). GFAP content did not vary

much when SGCs were activated by ATP. ET-1 treatment, on the other hand, caused a

decrease in GFAP levels (Figure 3 a and b). There was also a large variability in the data

between normalized values of GFAP across the two experiments (Figure 3 b). LPS

treatment apparently increased GFAP expression in the SGCs (Figure 3 c and d). This

data was somewhat consistent upon repetition and thus, LPS was utilized to further test

if it really activates SGCs.

The same protocol was followed and 3 replicates of Control and LPS treatment used

(Figure 3 c). GFAP content was normalized over the loading control (actin). The average of the replicates was calculated for each condition (Control and LPS treatment). This value was expressed as fold change by dividing it by control. The fold change across two such independent experiments was averaged and plotted on graph (Figure 3 d). The data shows no significant increase in GFAP content upon LPS treatment.

Figure 3. GFAP expression in SGCs in response to the activators – ATP, ET-1 and LPS(a) Activation of satellite glial cells by ATP (100µM), ET-1 (200nM) and LPS (1µg/mL). The treatment lasted 4 days and the modulators were replenished every alternate day. The cell lysates were subjected to Western Blot for glia activation marker GFAP. Actin was used as the loading control. (b) GFAP was quantified using BioRad Gel Doc system. The band intensity was normalized by actin and expressed as fold change with respect to GFAP levels in the control condition. Average values of 2 independent experiments were plotted on graph and expressed as mean ±SE. LPS treatment shows promising result. (c) Activation of satellite glial cells by LPS (1µg/mL). 3 replicate each, of control and LPS treatment, were subjected to Western Blot for GFAP expression. (d) Again, quantification was performed using Gel Doc system, was normalized by actin and expressed as fold change. The average of fold change was calculated for Control and LPS. The same process was repeated in another independent experiment. Finally the average of the values across these two independent experiments was expressed as mean ±SE and plotted on graph. GFAP levels did not increase upon LPS treatment this time.

Either these modulators do not activate SGCs or the cells are already activated due to

stress involved in culturing. This is plausible as GFAP content of control condition (not

exposed to any modulators) was already high (Figure 3). Another reason could be the

nature of experimental material. The culture was established using neonatal animals and

possibly GFAP is one of the proteins which is expressed at higher level in that

developmental stage compared to adult animals. Lastly, it is possible that GFAP, though

a very good activation marker for astrocytes, is not a good activation marker for SGCs.

3. Cell proliferation may be a better marker for SGCs activation

Cell proliferation is yet another marker for glia activation [Jasmin et al., 2010]. To

address the reason for SGCs not being activated by various modulators (Figure 3), the

same activation protocol (Figure 1 b) was used and cell proliferation was assessed. At

the end of treatment, the cells were fixed and stained for Ki67, a cell proliferation

marker that labels mitotically active cell population. Observations were made using

Olympus inverted microscope under 60x oil immersion objective and appropriate

fluorescence filter (Figure 4.1). Using Velocity 6.0 software, 90 fields in each dish were

observed. The Ki67 positive cells were counted in each field. Total cell count in the same

field was determined by switching to DAPI filter and counting the DAPI nuclei.

Proliferation percentage for each sample dish was thus, calculated using the simple

formula

Proliferation % = ((total no. of Ki67 positive cells)/total cell count)*100

LPS, among various modulators, showed increase in cell proliferation rate (Figure 4.2 a).

To test if this increase was significant, the experiment (Figure 1 b) was repeated thrice using LPS as glia activator. Each independent experiment had three replicates of each condition (Control and LPS treatment). For each experiment, the average of the tree replicates was calculated. The average across the three independent experiments was expressed as mean ±SEM and plotted on graph (Figure 4.2 b). Unpaired student t test was used for statistical analysis. The increase in proliferation rate upon LPS treatment was found to be statistically insignificant (p=0.14).

Figure 4.1 Satellite glial cell proliferation in response to LPS. DAPI (in blue) was used to stain all cell nuclei and Ki67 (in green) to stain actively dividing cells. The images were taken using Velocity 6.0 under 60x oil immersion objective. The insets show DAPI alone and FITC alone images. (a) Control condition showed comparable cell count but apparently lesser Ki67 positive cells. (b) Cells were treated with LPS (1 µg/mL) for a duration of 4 days. LPS treated cells were apparently more proliferative

Figure 4.2 Quantification of SGC cell proliferation in response to LPS. Ki67 positive cells were counted across 90 fields in each dish under 60x oil immersion objective. Total cell count in the same fields was determined by counting DAPI nuclei which was used to calculate proliferation percentage. (a) Out of the three modulators (ATP, ET-1 and LPS), LPS showed increase in rate of cell proliferation (n=1). (b) The cultured cells were treated with LPS (1µg/mL). Three replicates of each condition (control and LPS) were maintained and average for each was calculated. The values plotted on the graph are mean ±SEM of data across three independent experiments. LPS induced increase in proliferation rate but the increase was not statistically significant (p=0.14).

Although the increase in the proliferation percentage did not reach statistical significance (p>0.05) across three independent experiments, the difference in proliferation percentage between the control and LPS treated samples still looks more consistent than change in GFAP expression assessed by western blot. Thus, it can be concluded that cell proliferation may be a better marker than GFAP expression for

assessing SGC activation in cell culture. More number of independent experiments

would give a better idea about the statistical significance of the data.

4. Neurotrophin expression upon SGC activation

SGCs express the neurotrophins NGF and BDNF (Figure 2). Since NGF and BDNF have

been described to be modulators of synaptic properties in the sympathetic nervous

system [Luther et al., 2013], it should be interesting to know how neurotrophin levels

are affected (if at all) upon SGC activation. To address this, the previously described

activation protocol was used (Figure 1 b) using LPS as activator at a concentration of 1

µg/mL. Cell lysates were used for western blot for NGF and BDNF. Actin was used as the

loading control. The level of these neurotrophins did not vary drastically upon LPS

treatment (Figure 5 a and c). The bands were quantified using BioRad Gel Doc system

and normalized by actin. Average of three replicates for each condition (Control and LPS)

was expressed as fold change by dividing it by control. Average of this fold change across

two independent experiments was plotted on graph (Figure 5 b and d). NGF levels

increase upon activation (Figure 5 b) while BDNF levels apparently downregulate upon

LPS treatment (Figure 5 d). There appears to be less variability in BDNF downregulation

across the two independent experiments compared to NGF upregulation (Figure 5 b and

d). More number of independent experiments could help determine the statistical

significance of these changes in neurotrophin levels.

Figure 5. Expression of NGF and BDNF in SGCs in response to LPS treatment (1 µg/mL). After LPS treatment, cell lysates were subjected to western blot for NFG and BDNF. Actin was used as loading control. Quantification was performed using BioRad Gel Doc system. Three replicates of each condition was maintained across two independent experiments and the average values were plotted on graph. (a) NGF expression in response to LPS treatment for three replicates each of control and LPS treated cells. (b) NGF levels were normalized by actin and expressed as fold change. Average across two independent experiments was expressed as mean ±stdev. (c) BDNF expression upon LPS treatment. Again, three replicates of each condition was maintained. (d) BDNF values were quantified and normalized by actin. These values were expressed as fold change. Average across two independent experiment was expressed as mean ±stdev.

5. Acute treatment of SGCs with LPS and Fractalkine

LPS activates astrocytes [Lin et al.,2008] but it did not significantly activate SGCs of the

sympathetic ganglia. It is possible that SGCs may be more reactive towards other

modulators. Fractalkine has been described to activate SGCs of sensory ganglia and

could be tested for SGCs of sympathetic ganglia. So far, SGC activation has only been

assessed in response to long-term stimulation. Another possibility is that the SGCs may

show an acute response to the modulators. This can be tested by short term treatment

of SGCs. To test both the hypothesis, the activation protocol was slightly modified

(Figure 1 c). While rest of the protocol remained the same, the treatment duration was reduced to hours as opposed to days. LPS (at different concentrations) and FRK were used to treat SGCs for 6 h, 12 h and 24 h (Table 2). Glia conditioned media was harvested and centrifuged at high speed to eliminate cell debris. The supernatants were then transferred to fresh tube. Since the TNF-α concentration range in the SGC samples was unknown, a set of samples - Control and FRK (6 h treatment), was concentrated 5 times to ensure TNF-α detection in case the TNF-α concentration in the sample falls within detectable limits. All samples were analyzed for TNF-α production using ELISA kit.

Standards and samples were loaded in duplicates. There was no apparent change in

TNF-α levels across samples (Figure 6).

Figure 6. TNF-α levels in SGC culture media in response to short term treatment with LPS and FRK. LPS and FRK were used to treat SGCs for a duration of 6h, 12h or 24 h. LPS was used at different concentrations: 0.5 µg/mL, 1 µg/mL or 2 µg/mL (Refer Table 2). SGC conditioned media was analyzed using ELISA kit to detect TNF-α production. (a) ELISA results. Standards were serially diluted and loaded in duplicates in wells 1 and 2 (A through H). Samples were loaded in the rest of the wells. The standards and TNF-α control show variation in TNF-α levels as indicated by change in color intensity. For the sake of clarity, color contrast was increased. The TNF-α did not vary across different samples as confirmed by readings from the ELISA plate (not shown) (b) Sample labels. C = Control, LPS = Lipopolysaccharide and FRK = Fractalkine. The wells are labelled for the activator used followed by duration of treatment (6, 12 or 24 hours). The concentration of LPS used is indicated within paranthesis (0.5, 1 or 2 µg/mL). FRK was used at a concentration of 100 ng/mL.

The fact that none of the samples produced change in color compared to standards, indicate that either the glia did not get activated or the SGCs got activated but the TNF-α was too diluted and below detectable limits. The latter is less likely as the samples concentrated 5 times (Figure 6; wells 5F, 6F, 5G and 6G) also did not show any increase in TNF-α levels upon concentration.

Discussion

Glia, in response to injury and infection, become reactive and their expression profile is significantly altered. They glutamate released by activated glia could significantly contribute to neuronal hyperexcitability and excitotoxicity observed in several neurological disorders.

Thus, glia may be attractive therapeutic targets for these neuropathological conditions

[Agulhon et al., 2012].

In vitro cell culture models offer a simple and easy way to test and study a hypothesis as well as also offer elucidation of the mechanism involved in a process [McMillan et al., 1994].

Thus, i tried to develop an in vitro model to study activation of SGCs in sympathetic ganglia.

The SGCs of sympathetic ganglia were treated with several activators of glia (ATP, ET-1, LPS and FRK). The activation of SGCS was assessed by western blot, immunocytochemistry and

ELISA, to check for activation markers such as GFAP, TNF-α and cell proliferation. There was no significant change in GFAP expression upon treatment with ATP, ET-1 and LPS (Figure 3).

However, LPS treatment (4 days) induced increase in cell proliferation. But this increase was statistically not significant (Figure 4.2). SGCs (in sensory ganglia) do not normally produce

TNF-α but in response to inflammatory molecules such as fractalkine, the expression of this secretary factor becomes several fold high [Souza et al., 2013]. The glia conditioned media

from SGC culture of sympathetic ganglia (treated with LPS and FRK) did not show TNF-α expression when analyzed by ELISA. Other SGC secretary factors such as NGF and BDNF showed variation upon LPS treatment but data from more number of experiments is needed to test statistical significance of this increase.

There are several possibilities for the reason for SGCs in my culture to be not activated. FRK is a chemokine released by neurons of sensory ganglia. There is increased production of FRK upon inflammation which activates SGCs of sensory ganglia (TNF-α production was used as the activation marker). Since SGCs of sympathetic ganglia also belong to peripheral nervous system, it was hoped that they react similarly to FRK and get activated, but they didn’t.

Either the activation protocol needs to be further optimized or FRK might not be a good activator for SGCs of sympathetic ganglia if they don’t express receptors for FRK like SGCs of the sensory ganglia.

Other reasons for SGCs not getting activated could be attributed to the activation markers assessed. Sometimes activation of glia may involve structural changes in GFAP rather than changes in expression levels. Thus, western blot and PCR cannot document activation when protein or RNA levels remain unaffected after activation [Norton et al., 1992 and Eng and

Ghirnikar 1994]. GFAP levels in cultured neonatal astrocytes were found to be several folds higher than the adult cells [Wu and Schwartz 1995]. This could be true for SGC culture as well. Thus, the reason for no significant change in GFAP levels (Figure 2) upon treatment

with activators could be explained by a possibility of already elevated GFAP expression in the neonatal animals. SGC culture from adult animals could test this hypothesis.

It is also possible that GFAP and TNF-α production may not be as good activation markers for SGCs of sympathetic ganglia as they are for astrocytes and SGCs of sensory ganglia.

Many researchers utilize cell proliferation as activation marker as well. For this study, cell proliferation was apparently a better SGC activation marker than GFAP (Figure 4). However, further optimization of protocol is needed to avoid variability in data.

Astrocytes exhibit slow and persistent activation in response to injury in vivo. GFAP levels remain at peak even 14 days post injury. [Ji et al., 2013]. However, it is possible that SGCs show an acute response to activation. Short term treatments of SGCs in culture (Table 2 and

Figure 6) did not show activation. However, it was a single experiment (n=1) and a single assay; and efficacy of short term treatment cannot be completely ruled out and need to be tested further.

The activation markers such as GFAP need careful assessment. Western blot is a semi- quantitative technique and cannot efficiently account for small variation in protein expression. Thus, a more sensitive quantitative approach such as ELISA was adopted to measure secretary activation markers such as TNF-α. This did not detect any TNF-α in any of the samples. Assuming, the SGCs did get activated, the reason for this result could be (i) activation did not lead to TNF-α production and other secretary factors such as NGF and

BDNF could be assessed if they could serve as activation markers (ii) the TNF-α production was below detectable limits in the SGC conditioned medium analyzed by ELISA. A sample set concentrated 5 times also showed negative results for TNF-α detection. Much highly concentrated samples could be analyzed next to check for TNF-α production. As mentioned earlier, activation of glia could result in structural changes in GFAP rather than variation in

GFAP expression [Norton et al., 1992 and Eng and Ghirnikar 1994]. In astrocytes, activation is commonly assessed by immonostaining GFAP fibres [Tran and Neary, 2006]. Thus, immunocytochemistry was used to analyze control and treated SGC cultures. The GFAP in these neonatal SGC cultures stained faintly with GFAP antibody and did not show any clear variation between control and treated samples (images not shown). The SGCs, however, stained well for proliferation marker Ki67 which was used to assess cell proliferation in these cells in response to activation.

Often, the activation of glia is mediated by other cell population such as neurons or microglia and in vitro systems, in such cases, are insufficient to establish and study activation of glia. [McMillan et al.,1994]. However, several glia population have been successfully activated in vitro using ATP [Gandelman et al., 2010], ET-1[Gadea et al., 2008],

LPS [Lin et al., 2008] and FRK [Souza et al., 2013] and the same may be expected of SGCs.

There is however, a need for a more optimized, SGC-specific activation protocol to achieve successful activation. As mentioned earlier, expression profile of neonatal and adult animals could significantly differ making it difficult to achieve glia activation; use of adult SGC culture could be the next best approach to successfully develop in vitro model for SGC activation in sympathetic system.

Also, it was established through this study that SGCs produce neurotrophins. Since these neurotrophic factors alter synaptic properties [Luther et al.,2013], it would be attractive to explore if the levels of these factors in SGCs change significantly in response to injury and what implication this has on diseases of the peripheral nervous system.

Bibliography

Agulhon C, Sun MY, Murphy T, Myers T, Lauderdale K, Fiacco TA. Calcium Signaling and

Gliotransmission in Normal vs Reactive Astrocytes. Front Pharmacol. 2012;3:139.

Elfvin LG, Björklund H, Dahl D, Seiger A. Neurofilament-like and glial fibrillary acidic protein-like immunoreactivities in rat and guinea-pig sympathetic ganglia in situ and after perturbation. Cell

Tissue Res. 1987 Oct;250(1):79-86.

Eng LF, Ghirnikar RS. GFAP and astrogliosis. Brain Pathol. 1994 Jul;4(3):229-37.

Gadea A, Schinelli S, Gallo V. Endothelin-1 regulates astrocyte proliferation and reactive gliosis via a JNK/c-Jun signaling pathway. J Neurosci. 2008 Mar 5; 28(10):2394-408.

Gandelman M, Peluffo H, Beckman JS, Cassina P, Barbeito L. Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis. J Neuroinflammation. 2010 Jun 9;7:33.

Gosselin RD, Suter MR, Ji RR, Decosterd I. Glial cells and chronic pain. Neuroscientist. 2010

Oct;16(5):519-31.

Govoni S, Pascale A, Amadio M, Calvillo L, D'Elia E, Cereda C, Fantucci P, Ceroni M, Vanoli E. NGF and heart: Is there a role in heart disease?.Pharmacol Res. 2011 Apr;63(4):266-77.

Hanani M. Satellite glial cells in sympathetic and parasympathetic ganglia: in search of function.

Brain Res Rev. 2010 Sep 24;64(2):304-27.

Jasmin L, Vit JP, Bhargava A, Ohara PT. Can satellite glial cells be therapeutic targets for pain control?. Neuron Glia Biol. 2010 Feb;6(1):63-71.

Ji RR, Berta T, Nedergaard M. Glia and pain: Is chronic pain a gliopathy?. Pain. 2013 Dec;154.

Lin ST, Wang Y, Xue Y, Feng DC, Xu Y, Xu LY. Tetrandrine suppresses LPS-induced astrocyte activation via modulating IKKs-IkappaBalpha-NF-kappaB signaling pathway. Mol Cell Biochem.

2008 Aug;315(1-2):41-9.

Lobsiger CS, Cleveland DW. Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease. Nat Neurosci. 2007 Nov;10(11):1355-60.

Lu H, Cheng PL, Lim BK, Khoshnevisrad N, Poo MM. Elevated BDNF after cocaine withdrawal facilitates LTP in medial prefrontal cortex by suppressing GABA inhibition. Neuron. 2010 Sep

9;67(5):821-33.

McMillian MK, Thai L, Hong JS, O'Callaghan JP, Pennypacker KR. Brain injury in a dish: a model for reactive gliosis. Trends Neurosci. 1994 Apr;17(4):138-42.

Miller G. Neuroscience The dark side of glia. Science. 2005 May 6;308(5723):778-81.

Norton WT, Aquino DA, Hozumi I, Chiu FC, Brosnan CF. Quantitative aspects of reactive gliosis: a review. Neurochem Res. 1992 Sep;17(9):877-85.

Poo MM. Neurotrophins as synaptic modulators. Nat Rev Neurosci. 2001 Jan;2(1):24-32.

Ridet JL, Malhotra SK, Privat A, Gage FH. Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci. 1997 Dec;20(12):570-7.

Souza GR, Talbot J, Lotufo CM, Cunha FQ, Cunha TM, Ferreira SH. Fractalkine mediates inflammatory pain through activation of satellite glial cells. ProcNatlAcadSci U S A. 2013 Jul

2;110(27):11193-8.

Takeda M, Takahashi M, Matsumoto S. Contribution of the activation of satellite glia in sensory ganglia to pathological pain. NeurosciBiobehav Rev. 2009 Jun;33(6):784-92.

Tran MD, Neary JT. Purinergic signaling induces thrombospondin-1 expression in astrocytes.

ProcNatlAcadSci U S A. 2006 Jun 13;103(24):9321-6.

Wu VW, Schwartz JP. Neurotrophic factor expression in cultured astrocytes from neonatal, adult and injured rat brain. Soc Neurosci Abstr. 1995; 21:1532.

Yao C, Yang D, Wan Z, Wang Z, Liu R, Wu Y, Yao S, Yuan S, Shang Y. Aspirin-triggered lipoxin A4 attenuates lipopolysaccharide induced inflammatory response in primary astrocytes.

IntImmunopharmacol. 2014 Jan;18(1):85-9.

Zhang H, Mei X, Zhang P, Ma C, White FA, Donnelly DF, Lamotte RH. Altered functional properties of satellite glial cells in compressed spinal ganglia. Glia. 2009 Nov 15;57(15):1588-99.

Zigmond RE, Vaccariello SA. Activating transcription factor 3 immunoreactivity identifies small populations of axotomized neurons in rat cervical sympathetic ganglia after transection of the preganglionic cervical sympathetic trunk. Brain Res. 2007 Jul 23;1159:119-23.