RESEARCH ARTICLE

Connexin43 Hemichannels MediateSecondary Cellular Damage Spread from the

Trauma Zone to Distal Zones in AstrocyteMonolayers

Maximiliano Rovegno,1,2 Paola A. Soto,3 Pablo J. S�aez,3 Christian C. Naus,4

Juan C. S�aez,3,5 and Rommy von Bernhardi1

The mechanism of secondary damage spread after brain trauma remains unsolved. In this work, we redirected the attentionto astrocytic communication pathways. Using an in vitro trauma model that consists of a scratch injury applied to an astrocytemonolayer, we found a significant and transient induction of connexin43 (Cx43) hemichannel activity in regions distal from theinjury, which was maximal �1 h after scratch. Two connexin hemichannel blockers, La31 and the peptide Gap26, abolishedthe increased activity, which was also absent in Cx43 KO astrocytes. In addition, the scratch-induced increase of hemichannelactivity was prevented by inhibition of P2 purinergic receptors. Changes in hemichannel activity took place with a particularspatial distribution, with cells located at �17 mm away from the scratch presenting the highest activity (dye uptake). In con-trast, the functional state of gap junction channels (dye coupling) was not significantly affected. Cx43 hemichannel activitywas also enhanced by the acute extracellular application of 60 mM K1. The increase in hemichannel activity was associatedwith an increment in apoptotic cells at 24 h after scratch that was totally prevented by Gap26 peptide. These findings suggestthat Cx43 hemichannels could be a new approach to prevent or reduce the secondary cell damage of brain trauma.

GLIA 2015;63:1185–1199Key words: traumatic brain injury, connexins, P2 receptors, astroglia, apoptosis

Introduction

Traumatic brain injury (TBI) is a leading cause of morbid-

ity and death, especially for people under 45 years of age.

In the United States, 1.4 million incidents of TBI occur

annually resulting in 235,000 hospitalization, 50,000 death,

and USD $60 billons in costs (Langlois et al., 2006).

TBI is characterized by a primary damage zone at the

impact site, which propagates to neighboring zones because

of ischemia, excitotoxicity, cellular tumefaction, and inflam-

mation (Werner and Engelhard, 2007). In the past decade, all

clinical trials designed to test possible neuroprotective proto-

cols have failed (Jain, 2008). Consequently, an effective neu-

roprotective drug is still lacking, mainly because relevant

target molecules have not been identified. These negative

results can be explained in part by a neuron-centered

approach, which could lead to overlooking the participation

of other cell types and pathogenic mechanisms (Rovegno

et al., 2012). Alternatively, cell types that could play a rele-

vant role in neuronal survival are glia and particularly astro-

cytes, because they play major roles both in physiologic and

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22808

Published online March 2, 2015 in Wiley Online Library (wileyonlinelibrary.com). Received June 10, 2014, Accepted for publication Feb 5, 2015.

Address correspondence to Maximiliano Rovegno, Departamento de Medicina Intensiva, Facultad de Medicina, Pontificia Universidad Cat�olica de Chile, Marcoleta

367, Santiago, Chile. E-mail: maxrovegno@uc.cl

From the 1Laboratorio de Neurociencias, Departamento de Neurolog�ıa, Facultad de Medicina, Pontificia Universidad Cat�olica de Chile, Santiago, Chile; 2Departa-

mento de Medicina Intensiva, Facultad de Medicina, Pontificia Universidad Cat�olica de Chile, Santiago, Chile; 3Departamento de Fisiolog�ıa, Facultad de Ciencias

Biol�ogicas, Pontificia Universidad Cat�olica de Chile, Santiago, Chile; 4Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British

Columbian, Vancouver, British Columbia, V6T 1Z3, Canada; 5Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valpara�ıso, Valpara�ıso, Chile

The data from this study were presented by Maximiliano Rovegno as partial fulfillment of the requirements to obtain the PhD degree in Medical Sciences at the

Pontificia Universidad Cat�olica de Chile.

Additional Supporting Information may be found in the online version of this article.

VC 2015 Wiley Periodicals, Inc. 1185

pathologic states (Barres, 2008). Astrocytes are highly inter-

connected via gap junctions that allow for spatial buffering of

extracellular K1, H1, and glutamate, among others (Fields

and Stevens-Graham, 2002). Moreover, astrocytes express

receptors for a wide variety of neurotransmitters and release

diverse neurotransmitters capable of regulating functions of

nearby cells, a property knows as gliotransmission (Bezzi and

Volterra, 2001). Gap junctions are clusters of intercellular

connexin-based channels that facilitate several of the afore-

mentioned astroglial functions (Theis and Giaume, 2012).

Connexins are transmembrane proteins that form two

different pathways for intercellular communication. One

pathway involves the gap junction channels (GJCs) located

between adjacent cells allowing direct cell-to-cell transfer of

ions and small molecules. The other pathway is through hem-

ichannels (HCs, half GJCs) located at unopposed cellular sur-

face that permit the exchange between intra- and extracellular

compartments, including release of autocrine/paracrine signal-

ing molecules (e.g., ATP, NAD1, and glutamate) to the

extracellular milieu (S�aez et al., 2003). In vivo, astrocytes

express connexin43 (Cx43) and lower levels of Cx26 and

Cx30 (Nagy et al., 1999, 2001), whereas in culture they

express only Cx43 (Dermietzel et al., 1991; Giaume et al.,

1991). In addition, astrocytes express pannexin1 (Panx1)

(Iglesias et al., 2009; Suadicani et al., 2012), a member of a

family of three glycoproteins (Penuela et al., 2009) that also

form HCs permeable to small molecules such as ATP (Che-

keni et al., 2010; Huang et al., 2007b;).

In stroke, a possible role of astrocytes contributing to

the amplification of damage has been previously proposed

(Rossi et al., 2007). In fact, they are the principal defense

against excitotoxicity by active clearance of glutamate (Roth-

stein et al., 1996). However, energy failure could reverse

transport (Allen et al., 2004) and subsequently result in

down-regulation of the glutamate transporter GLT-1 (Rao

et al., 2001), which exacerbates excitotoxicity. Under tissue

stress, astrocytes could spread apoptotic signals through GJCs

(Lin et al., 1998) and HCs (Orellana et al., 2011a,2011b). In

stress induced by metabolic inhibitors, the enhanced connexin

HC activity of astroglia accelerates cell death (Contreras

et al., 2002).

In a model of trauma in vivo, increased Cx43 immunore-

activity is observed in the ipsilateral hippocampus 24–72 h post

injury (Ohsumi et al., 2010), and phosphorylation of Cx43 is

detected already at 1 h post-trauma (Ohsumi et al., 2006). In

hippocampal slices, the connexin-based channel blockers, car-

benoxolone and octanol, significantly reduced cell death post

trauma (Frantseva et al., 2002). Similar results were obtained

using antisense oligonucleotides for Cx43, and in brain slices

from Cx43 KO mice (Frantseva et al., 2002). However, these

studies did not elucidate the relative importance of GJCs and

HCs and the molecular mechanism that regulates the intercel-

lular communication pathways formed by connexins in TBI.

Notably, a previous study in spinal cord trauma suggested the

participation of Cx43 HCs in early damage spread. In an ex

vivo model of spinal cord trauma, the use of peptide5, a specific

mimetic peptide against the second loop of Cx43, prevented

edema, astrogliosis, and neuronal death after injury (O’Carroll

et al., 2008). The selective inhibition of the HC activity was

correlated with the peptide concentration and its protective

results (O’Carroll et al., 2008).

Among tissue changes that occur post TBI in vivo, there

is a rapid and transitory increase in extracellular K1 concen-

tration, ([K1]e), to near 55 mM (Nilsson et al., 1993), which

occurs in hyperosmolar conditions measured in contuse brain

areas in clinical and experimental settings (Kawamata et al.,

2007). This abrupt increase in [K1]e can open Cx50 HCs

and Panx1. In fact, replacement of extracellular Na1 by K1

induces a >10-fold potentiation of Cx50 HCs currents (Sri-

nivas et al., 2006). Similarly, high extracellular K1 concentra-

tions can induce opening of Panx1 (Jackson et al., 2014;

Silverman et al., 2009). However, the effect of high [K1]e on

Cx43 HCs has not been evaluated.

Here, we used an in vitro model of trauma by

scratching a monolayer of cortical rat astrocytes as previ-

ously described (Lau and Yu, 2001; Tecoma et al., 1989;

Udawatte and Ripps, 2005) and studied the connexin-

based channel pathway involved in damage propagation.

We found a spatial distribution of increased HC activity:

cells at �17 mm away from the scratch gained Cx43 HC-

mediated communication without changes in GJC-

dependent coupling. This increase in HC activity was

inhibited by connexin HC blockers, was potentiated by

60 mM K1e, was prevented by blocking P2 purinergic

receptors, and did not occur in astrocytes from Cx43 KO

mice. The increased HC activity was associated with an

increment of apoptotic cells at 24 h after scratch, which

was abolished by inhibition of connexin HC.

Materials and Methods

ReagentsApyrase VI–VII, ATP bioluminescent assay kit, brilliant blue G

(BBG), bovine trypsin, EDTA, ethidium (Etd) bromide, LaCl3, lido-

caine, Lucifer yellow (LY), poly-L-lysine, suramin, oxidized ATP

(oATP), and ruthenium red (RuR) were purchased from Sigma-

Aldrich (St. Louis, MO). Gap26 and 10panx1 were synthetized by

SBS Genetech (Beijing, China). Dulbecco’s modified Eagle’s/F12

medium (DMEM/12; 1:1), fetal bovine serum (FBS), and penicillin/

streptomycin were obtained from Invitrogen (Grand Island, NY).

Annexin V Alexa-488, and propidium iodide (PI) were purchased

from Molecular Probes (Eugene, OR). ATP-g-S tetra lithium salt

was obtained from Roche (Indianapolis, IN) and staurosporine from

Cell Signaling (Danvers, MA). Rat Tumor necrosis factor alpha

1186 Volume 63, No. 7

(TNF-a) and Interleukin-1 beta (IL-1b) were purchased from eBio-

science (San Diego, CA).

Drugs and Stock SolutionsArtificial cerebrospinal fluid (aCSF, in mM: 154 NaCl, 5.4 KCl,

1.0 MgCl2, 2.3 CaCl2, 2.5 HEPES, and 5 glucose, pH 7.42 in

ultrapure water (W3500; Sigma-Aldrich)) was used as recording

solution and for performing the trauma protocol. Etd for the dye

uptake experiments was prepared as a 25 mM stock solution in

water and diluted to 5 lM in aCSF before applying it to cells. The

18b-glycyrrhetinic acid was prepared in ethanol as a 3500 stock

solution. BBG, LaCl3, and oATP were dissolved in aCSF at 3100

stock solution. Suramin was prepared as a 200 lM solution in

aCSF. Staurosporine was prepared as a 31,000 stock solution in

dimethyl sulfoxide (DMSO). All aqueous stock solutions were

diluted to their final concentrations in sterile aCSF.

AnimalsNewborn (P2) Sprague-Dawley rats were obtained from the animal

facility of the Pontificia Universidad Cat�olica de Chile. In addition,

Cx43-deficient astrocytes (Cx432/2) were obtained from constitutive

Cx43 knock-out mice (Reaume et al., 1995), whereas Cx431/1

wild-type (WT) astrocytes were cultured from mice with the same

genetic background. All experimental protocols were approved by

the Ethical and Well Being Animal Committee of the Medical

School from the Pontificia Universidad Cat�olica de Chile (CEBA

09-034).

Cell CulturesBriefly, meningeal tissue was removed, and the neocortex was minced

and incubated at 37�C for 10 min in Ca21-free Hanks’ solution

containing (0.25%) bovine trypsin and 5 mM EDTA. Then, tissue

was mechanically dissociated. Cells were seeded in 75-cm2 culture

flaks coated with poly-L-lysine (one brain per flask), supplemented

with complete medium (DMEM-F12, 10% FBS, 1% penicillin–

streptomycin) and incubated at 37�C in a water-saturated atmos-

phere with 5% CO2. After �21 days in culture, flasks were treated

with 12 mM lidocaine and incubated at 37�C for 10 min to detach

microglial cells. The supernatant was discarded, and the remaining

cells were recovered by trypsination and shaking for 10 min. To

increase the purity of astrocyte cultures, a pre-plating step was

added. Cells were transferred to 94-mm diameter bacteriological

Petri dish (Sterilin, Newport, United Kingdom) and pre plated for

1 h. Unattached cells were seeded at 4 3 105/dish into 35-mm

diameter plastic dishes (Greiner, Frickenhausen, Germany) contain-

ing or not, 12 mm-diameter glass coverslips, 3 per dish (Marienfeld,

Lauda-K€onigshofen, Germany). Finally, cells were grown to conflu-

ence (�2 weeks), and all medium was changed every 72 h until the

experiments were performed.

Trauma ProtocolAstrocyte enriched cultures were subjected to trauma-like conditions

in aCSF medium. Conditions were as follows, Control: aCSF; K1:

aCSF containing 60 mM KCl; Trauma: single scratch with a G25 nee-

dle across the cell monolayer; TK1: scratch trauma performed in

aCSF containing 60 mM KCl. After 2 min incubation, cultures were

washed once with fresh aCSF. Then, cultures were placed back into

the incubator with their previous culture medium, at 37�C in a water

saturated atmosphere with 5% CO2, until the recording time.

Dye Uptake and Time-Lapse Fluorescence ImagingCells plated into 35-mm diameter dishes were washed once with

aCSF and exposed to 5 lM Etd in aCSF. The fluorescence intensity

was recorded for nuclei of 30 cells in regions of interest, with a

water immersion lens in an Olympus 51W1I upright microscope

(Melville, NY). Images were captured with a Q Imaging model

Retiga 13001 fast cooled monochromatic digital camera (12-bit;

Qimaging, Burnaby, BC, Canada) every 20 s (exposure time 5 30

ms, gain 5 0.5) and Metafluor software (version 6.2R5; Universal

Imaging, Downingtown, PA) was used for image analysis and fluo-

rescence quantification. For data representation and Etd uptake rate

(slope) calculation, the average of two independent background

intensity measurements (FB, expressed as arbitrary units or AU) was

subtracted to the fluorescence intensity of cells at each time interval

(F1). Results of this calculation (F1 2 FB) at each time interval for

each of 30 cells were averaged and plotted against time (expressed in

minutes) during 20 min. Etd uptake rates were calculated using

Microsoft Excel software (Redmond, W) and expressed, as AU/min.

Microscope and camera settings remained unchanged in all

experiments.

Dye CouplingThe intercellular communication via GJCs was evaluated by ionto-

phoretic injection of single cells with 5% wt/vol LY (MW 5 457.24,

21) in 150 mM LiCl through glass microelectrodes, as previously

described (Corval�an et al., 2007). Briefly, astrocyte enriched cultures

were visualized in an inverted microscope (TE 200; Nikon, Melville,

NY) equipped with a xenon arc lamp and filters for LY (excitation

wavelength 450–490 nm; emission wavelength above 520 nm). Two

min after microinjecting dye into one cell, surrounding cells were

examined to determine whether dye transfer to them has occurred.

The incidence of dye coupling was calculated as the percentage of

injected cells in which the dye transferred to at least one adjacent

cell, and the index of dye coupling corresponded to the number of

cells to which dye spread in positive cases. In all experiments, the

incidence of dye coupling was evaluated by injecting a minimum of

10 cells.

ImmunofluorescenceCells plated onto coverslips were washed in phosphate-buffered

saline (PBS) with 0.1 mM Ca21 and fixed in fresh 4% p-formalde-

hyde at room temperature (�21�C) for 30 min. Then, cells were

incubated for 30 min at 4�C with blocking solution containing

10% goat serum in PBS, and after several washes with PBS, they

were incubated with isolectin GS-IB4 Alexa-568 (1:100; Molecular

Probes, Eugene, OR) or polyclonal rabbit anti-GFAP (1:400; Dako,

Glostrup, Denmark). Coverslips were washed in PBS and incubated

at room temperature for 2–4 h with goat anti-rabbit IgGs conju-

gated to Alexa 488 (1:100 in blocking solution; Molecular Probes).

Coverslips were washed in PBS and nuclei were stained with 0.1

Rovegno et al.: Astroglial HCs and Damage Propagation in Trauma

July 2015 1187

lg/mL Hoechst 33258 (B2883; Sigma-Aldrich) diluted in water.

Final washes were performed with PBS followed by one wash with

distilled water. Samples were mounted in fluorescence mounting

medium (Dako). Images were obtained with an inverted fluores-

cence microscope (DMIL, Leica, Wetzlar, Germany) equipped with

a color digital camera. Cell identity was quantified in 10 fields per

coverslip using the NIH ImageJ program.

Apoptosis QuantificationCoverslips with cell monolayers were washed in PBS at 4�C. Then, cells

were incubated with 5 lM annexin V Alexa-488 and 20 lg/mL PI in

buffer binding solution (in mM: 10 HEPES, 140 NaCl, 2.5 CaCl2, pH

7.4), at room temperature for 15 min. Cells were washed twice with

buffer binding solution and fixed in fresh 4% formaldehyde at 21�C for

15 min. Then, coverslips were washed several times with PBS, stained

with Hoechst, and mounted as described in the immunofluorescence

section. Photomicrographs were obtained with an epifluorescence Olym-

pus Bx51 microscopy (Melville, NY) equipped with a color digital cam-

era. Apoptotic cells were positive for annexin V and negative for PI. Ten

fields were quantified per condition for each independent experiment,

using the NIH ImageJ program. Additionally, apoptosis was assessed by

detecting the presence of cleaved caspase-3 by Western blot analysis. Cell

cultures were washed three times with PBS at 4�C and harvested by

scraping with a rubber policeman and homogenized in loading buffer,

electrophoretically separated on a 10% polyacrylamide gel, and trans-

ferred to a nitrocellulose membrane (0.45 lm, BioScience, Rockaway,

NJ). The membrane was incubated with blocking solution (5% nonfat

milk, 0.1% Tween-20 in tris-buffered saline (TBS)) at 4�C for 4 h. The

blot was probed with polyclonal rabbit antibody recognizing cleaved

caspase-3 (Asp 175, a 17/19 kDa protein) (1:1,000, Cell Signaling, Dan-

vers, MA), diluted in 5% wt/vol nonfat dry milk, 13 TBS, 0.1%

Tween-20 at 4�C with gentle shaking overnight. Later, the membrane

was incubated with the secondary antibody, a horseradish peroxidase-

conjugated goat anti-rabbit IgG (1:2,000, Santa Cruz, CA). Proteins

were visualized by chemiluminescence (kit SuperSignal; Thermo Scien-

tific, Rockford, IL), and scanned (Quansys scanner; BioScience, West

Logan, UT).

Determination of Nitrites (NO22)

Cell cultures were exposed to the various experimental conditions.

NO22 secreted to the culture medium was determined by the

Griess assay (Pfeiffer et al., 1997). In brief, 50 lL of medium

was mixed with 10 lL ethylendiaminetetraacetic acid (EDTA)/

H2O 1:1 (0.5 M, pH 8.0) and 60 lL of freshly prepared Griess

reagent (20 mg N-[1-naphtyl]-ethylendiamine and 0.2 g sulfanila-

mide dissolved in 20 mL of 5% phosphoric acid, wt/vol). Stand-

ard curves were established with 1–80 lM NaNO2. Absorbance

was measured at 570 nm in a microplate auto reader (ANTHOS

2010, Anthos Labtec Instrument, Eugendorf, Austria).

ELISA of TNF-aThe TNF-a concentration in extracellular medium was determined by

sandwich ELISA (eBioscience, USA). For the assay, 100 lL of culture

supernatants were added to an ELISA plate well and incubated at 4�C

overnight. The detection antibody was incubated at room temperature

for 1 h and the reaction was developed with avidin–HRP and substrate

solution. The plate was read at 450 nm with reference to 570 nm using

a microplate auto reader (ANTHOS 2010, Anthos Labtec Instrument,

Eugendorf, Austria). For quantification, samples readings were interpo-

lated in a standard curve with a range of 8–2,000 pg/mL.

ATP AssayThe ATP released to the culture medium was measured after trauma

in vitro using bioluminescence (Luciferin–firefly luciferase reaction

kit; Sigma-Aldrich, St. Louis, MO). In brief, samples of extracellular

medium were mixed with ATP assay according to manufacturer’s

protocol, and the resulting bioluminescence was immediately meas-

ured with a microplate auto reader (Synergy HT Multi-Mode,

Winooski, VT). Readings were interpolated in a standard curve with

a range of 10213210210 M ATP.

Potassium Ion Measurement[K1]e was studied in culture medium after trauma in vitro, using the

potassium ion selective electrode technique in a commercial reader

(Cobas 6000, Indianapolis, IN).

Statistical AnalysisData are expressed as mean 6 SEM. The statistical analyses were per-

formed using GraphPad Prism software. Effects on dye coupling and

dye uptake were analyzed by one- or two-way ANOVA, followed by a

multiple comparisons Dunnett’s test or the Bonferroni correction,

respectively. For dye coupling studies, basal values and those obtained

after treatment with a drug were compared with the Wilcoxon matched

pairs test. Lineal regression was used to test association between varia-

bles, often by Pearson correlation. Rho’ Spearman correlation was pre-

ferred in case of ordinal versus continuous variables association. In all

cases, a P value less than 0.05 was considered statistically significant.

Results

Characterization of the in Vitro Trauma ModelMechanical injury on cells can be produced by several

approaches including transection, compression, or stretching

(Kumaria and Tolias, 2008). Whereas cell stretch injury is a

suitable model of diffuse trauma (Ellis et al., 1995), scratch

injury is a model of focal trauma based on physical disruption

of cells (Tecoma et al., 1989). Previous studies using the

scratch trauma model have reproduced excitotoxicity (Tecoma

et al., 1989), inflammation (Lau and Yu, 2001), and apopto-

sis (Udawatte and Ripps, 2005), all important components of

TBI (Werner and Engelhard, 2007).

The composition of glial cell cultures was assessed by

immunostaining with astrocyte and microglia markers (Glial

fibrillary acidic protein (GFAP) and Isolectin B4, respec-

tively). We found that 93.3 6 1.5% cells were GFAP positive

(astrocytes) and a 6.7 6 1.5% were Isolectin B4 positive

(microglia) (Supp. Info. Fig. S1A,B).

The scratch applied to the astrocyte monolayer was

highly reproducible, leaving a wound �60 mm width

1188 Volume 63, No. 7

(Fig. 1A,B and Supp. Info. Fig. S2A). After the scratch, cul-

tures were kept at 37�C in the incubator for 1 h before their

assessment. We chose this time frame because previous studies

of trauma in vitro demonstrated early changes such as the

acute and transitory elevation of the [K1]e within minutes

(Nilsson et al., 1993), as well the induction of Cx43 phos-

phorylation (Ohsumi et al., 2006) and increment of cytokines

at �1 h after scratch (Lau and Yu, 2001).

Trauma in Vitro Increases Cx43 HC Activity inEnriched Astrocyte MonolayersBecause connexin-based channels permit physical contact

between the cytoplasm of cells (GJCs) or with the extracellu-

lar space (HCs), they allow the generation of two different

space dye patterns in experiments using extracellular dye: (1)

concentration gradient of dye spread from the wound area

toward the monolayer interior via GJCs, and (2) cell clusters

showing dye uptake due to HC opening in foci located dis-

tantly from the scratch region, as described previously (Reta-

mal et al., 2007). The dye stain pattern was evaluated in

three regions of interest (ROI), at different distances from the

scratch: “center” at �200 mm, “middle” at �8 mm, and

“periphery” at �17 mm, (Supp. Info. Fig. S2B).

The effect of scratch on the activity of HCs was meas-

ured by Etd uptake. Astrocytes under Control conditions

showed a low Etd uptake (Fig. 1B,D–F), as previously

described (Orellana et al., 2010; Retamal et al., 2006; S�aez

et al., 2013). One-hour after scratch, cell clusters located

away from the scratch showed a 1.6-fold increase of Etd

uptake (Fig. 1A–F and Supp. Info. Fig. S3A). Notably, the

addition of 60 mM K1e at the time of scratch (hyper-osmo-

lar TK1 condition) further increased Etd uptake at peripheral

ROIs, up to 2.0-fold of the control value (Fig. 1B–F). Also,

the addition of 60 mM K1e without scratch (hyper-osmolar

K1 condition) resulted in a tendency of increased Etd uptake

(Fig. 1E and Supp. Info. Fig. S3A). Thus, induction of Etd

uptake profile was observed across the following conditions:

TK1>Trauma>K1>Control (Fig. 1F). In time course

experiments, the increment in dye uptake was evident at �30

min, and was maximal at �1 h after scratch (Supp. Info. Fig.

S3B), whereas dye uptake was not significantly increased at

3 h after scratch, except in control cultures, suggesting that

manipulation of control cultures induced HCs activity. In all

conditions studied, including Control, Etd uptake was drasti-

cally reduced by 200 lM La31, a connexin HC blocker (Fig.

1D,E). As aforementioned, the dye uptake detected in control

cultures inhibited by La31 was considered as basal HC

activity (Orellana et al., 2010; Retamal et al., 2006; S�aez et

al., 2013). The application of 50 lM 18b-glycyrrhetinic acid

(b-GA), a connexin GJC/HC blocker, further decreased the

Etd uptake to values below basal condition (Fig. 1E).

The only connexin expressed by cortical astrocytes in

culture is Cx43 (Dermietzel et al., 1991; Giaume et al.,

1991). However, cell cultures used in the present work con-

tained �7% microglia, which under resting conditions could

express Cx36 and Cx45 (Dobrenis et al., 2005). Moreover,

astrocytes also express Panx1 (Huang et al., 2007a; Iglesias

et al., 2009; Jackson et al., 2014; Suadicani et al., 2012) that

may form functional channels under the conditions studied.

Thus, to determine whether only Cx43 HCs mediate the

scratch-induced cell permeabilization, we used specific

mimetic peptides: Gap26 and 10panx1, which act at the level

of the first extracellular loop of Cx43 and Panx1, respectively

(Evans et al., 2006; Pelegrin and Surprenant, 2006). Both

peptides were used at 150 lg/mL and were applied 10 min

before recording. Only Gap26 prevented the increase in Etd

uptake induced by Trauma or TK1 (Fig. 1E and Supp. Info.

Fig. S4A,B). Furthermore, cells exposed to Gap26 achieved a

similar Etd uptake to that observed after acute blockade with

La31 (Fig. 1E), indicating that Cx43 HCs mediated the

Trauma or TK1-induced cell permeabilization. In contrast,10panx1 did not affect the Etd uptake induced by Trauma or

TK1 (Fig. 1E). This, in addition to the La31 effect that

blocks connexin HCs but not Panx1 channel (Pelegrin and

Surprenant, 2006), excluded the possible participation of

Panx1 channel. Also, the participation of the recently

described channel CALMH1 permeable to ions and small

molecules (Taruno et al., 2013) was discarded using 20 lM

ruthenium red (RuR), that decreased by 19% Etd uptake for

both the Control and TK1 condition (Supp. Info. Fig.

S4C,D). To further confirm this interpretation, Cx43 KO

astrocytes were used. Astrocytes from Cx432/2 mice were

compared with their WT counterpart (Cx431/1). Only WT

astrocytes showed Etd uptake, with a similar profile to that of

rat astrocytes (Fig. 1G). In fact, Etd uptakes of Cx432/2

astrocytes were lower than that of control Cx431/1, support-

ing the existence of Etd uptake due to a basal Cx43 HC

activity.

As previously mentioned, the magnitude of b-GA-

induced blockade was more robust than that induced by

La31 at every condition (Fig. 1E). Consequently, we decided

to study the possible contribution of GJCs in the scratch-

induced dye uptake.

The Intercellular Coupling via GJCs is not Affectedin Astrocyte Cultures at 1 Hour After ScratchPrevious work on trauma in hippocampal slices showed a cor-

relation of the functional presence of GJCs with spread of

cell death, but the participation of HCs was not evaluated

(Frantseva et al., 2002). We studied whether GJCs were also

affected in the model of trauma in vitro used hereby quantify-

ing transfer of the dye LY between cells.

Rovegno et al.: Astroglial HCs and Damage Propagation in Trauma

July 2015 1189

FIGURE 1: Trauma and Trauma plus 60 mM [K1]e increases Cx43 HC activity at peripheral ROIs. One hour after the experimental condi-tions, astrocyte cultures were incubated with 5 lM Etd. Fluorescence was measured every 30 s [518 nm, arbitrary units (A.U.) of fluores-cence intensity], n 5 30 cells per experiment. In representative photomicrographs the exposure time of dye was 10 min. A: Panoramicscratch Etd uptake and bright field microscopy from the injury site to periphery, bar 5 1 mm. Etd uptake was increased at peripheralregion of the scratch at 1 h, with a “cluster” distribution. B: Etd uptake (snap-shots) and bright fields of defined ROIs, bar 5 50 lm. Etduptake was increased post Trauma, compared with Control and was potentiated in the TK1 condition. A, B: Arrowheads mark scratchsites. C: Normalized number of Etd fluorescent cells by subtracting the fluorescent cells at the Control condition. In the peripheryoccurred an increment of Etd fluorescence, and comparing to Trauma, there was a greater increase in TK1 condition, with 1.8-fold morefluorescent cells at periphery. D: Representative time-lapse measurement of Etd uptake under Control, Trauma, and TK1 conditions atthe periphery. Etd uptake was induced by scratch; the effect was potentiated by K1, and was reduced by 200 lM La31. E: Average dyeuptake. In peripheral ROIs, Trauma and TK1 increased Etd uptake 1.6- and 2.0-fold compared with controls, respectively. This incrementwas abolished by 200 lM La31, 50 lM b-GA, or 150 lg/mL Gap26 extracellular peptide. The blocking of Panx1 with 10panx1 peptidehas no effect. Each plotted value represents the mean 6 SEM of 10 independent experiments (*= P < 0.05; **= P < 0.01; ***= P < 0.001).Conditions were as indicated in (G) using same label code. F: Average dye uptake versus conditions arranged according to their injurypotential (i.e., Control< K1< Trauma< TK1). There was a significant correlation between the Etd uptake and the injury conditions. (Rho’Spearman correlation P < 0.01). Plotted data is from values shown in panel (E). G: Astrocytes cultured from Cx432/2 mice were com-pared with wild-type counterparts. There was a similar increment in Etd uptake after Trauma and TK1 condition in Cx431/1 astrocytes,but not in astrocytes from Cx432/2 mice. Each plotted value represents the mean 6 SEM of 3–5 independent experiments.

Since leakage of microinjected LY might occur through

HCs, dye coupling was evaluated in the presence of 200 lM

La31 to block HCs. Extracellular La31 did not modify the

intercellular transference of microinjected LY in Control con-

ditions (Supp. Info. Fig. S6A,B). Notably, Trauma or TK1

condition (Fig. 2A–C) did not affect coupling between astro-

cytes. In fact, no changes were noted in the index or inci-

dence of coupling of any studied condition at peripheral

ROIs (Fig. 2A–C), or other regions (data not show). This

information together with the Etd uptake induction profile

inhibited by La31 and b-GA, indicate negligible GJCs contri-

bution to the scratch-induced dye uptake. Thereby, the La31

sensitive component seems to be responsible for the Etd

uptake increment in Trauma and TK1 conditions.

Because Gap26 may also block GJCs (Evans et al.,

2006), it was necessary to revaluate the cell coupling state.

We studied LY transfer in the presence or absence of Gap26

peptide for 10 min. After 1 h of Gap 26 application, dye

coupling remained as in control cells (Fig. 2D,E), supporting

the inference that only Cx43 HCs mediated the Etd uptake

induction profile observed in all conditions.

Elevated Extracellular K1 and Activation of a P2Receptor-Dependent Pathway MediateScratch-Induced Increase in Cx43 HC ActivityTo further examine the mechanism underlying the increase in

Cx43 HC activity induced by scratch, we evaluated the effect

of elevated extracellular K1 concentration. In our model, an

increase of [K1]e after scratch was not observed (Supp. Info.

Fig. S5A). However, after in vivo trauma there is an acute

and transitory increase in [K1]e reaching a concentration of

55 mM associated with tissue hyper-osmolarity (Kawamata

et al., 2007; Nilsson et al., 1993;). This K1 concentration

could facilitate opening of Cx50 HCs, in an iso-osmolar con-

dition (replacement of extracellular Na1 with K1) (Srinivas

et al., 2006). Therefore, we decided to study the acute effect

of high [K1]e and extracellular osmolarity on the Etd uptake

FIGURE 2: Trauma does not affect the cell–cell coupling of astrocytes. Lucifer yellow (LY) transfer was examined after 2 min of dyemicroinjection, 1 h after the experimental conditions. A: Representative photomicrographs of dye coupling with their correspondingbright fields, bar 5 50 lm. The (*) denotes the cell microinjected with LY. B: Index 5 absolute number of cells to which the dye was trans-ferred. C: Dye coupling incidence 5 percentage of cases in which the dye transferred to at least one adjacent cell. The cells remainedcoupled after the diverse conditions studied, at 1 h. D, E: Index and dye coupling incidence after 1 h incubation with 150 lg/mL Gap26. The concentration and time of application of Gap 26 used in the study of Cx43 HC activity did not change the cell-cell couplingbetween astrocytes. Each plotted value represents the mean 6 SEM of 10 independent experiments (n.s., not significant difference).

Rovegno et al.: Astroglial HCs and Damage Propagation in Trauma

July 2015 1191

of astrocyte monolayers. To manipulate the [K1]e and osmo-

larity separately, we compared the effect of an extracellular

solution with 60 mM K1 (addition of KCl) versus a solution

where 60 mM Na1 was replaced by 60 mM K1 (iso-osmo-

lar). Moreover, a saline solution with additional 60 mM

sucrose was used to assess a pure hyper-osmolar effect. All

experiments were performed in a superfusion chamber for

rapid exchange of extracellular solutions (< 1 min). For each

experiment, the basal value was obtained in recording solu-

tion followed by the modified extracellular solution.

In cultures exposed to hyper-osmolar 60 mM [K1]e

aCSF, the Etd uptake increased to 1.5-fold. Similarly, aCSF

containing 60 mM sucrose also increased the Etd uptake to

1.9-fold of basal value. However, the greatest induction of

Etd uptake was observed with iso-osmolar 60 mM [K1]e,

reaching a 2.3-fold increase of the basal value (Fig. 3A,B).

Finally, to further differentiate the osmotic effect of [K1]e

from its ionic effect, we used aCSF in which 60 mM Na1

were replaced with 60 mM K1 and 60 mM sucrose were

added. This hyper-osmolar solution did not increase Etd

uptake beyond that observed with 60 mM sucrose (Fig. 3A).

This last finding, together with the most robust induction of

Etd uptake with replacement of Na1e by K1

e, suggest a

direct and rapid effect of K1 ions on Cx43 HC activity.

Then, K1 ion direct effects were evaluated with a concentra-

tion–response curve for hyper-osmolar high [K1]e aCSF and

Etd uptake. From 5.4 mM to 120 mM KCl, there was a lin-

ear relationship between [K1]e and connexin HC activity

(Fig. 3C).

Extracellular ATP plays important modulatory functions

in physiologic and pathologic states (Burnstock, 2008). It can

mediate neuron and astrocyte death in vitro and in vivo(Amadio et al., 2002; Franke et al., 2009; Jackson et al.,

2014; Jeong et al., 2010). The extracellular ATP concentra-

tion rises after trauma induced by moderate cell stretch in

primary astrocyte cultures (Ahmed et al., 2000), and can per-

sist elevated for �1 h (Neary et al., 2005). In our study,

extracellular ATP increased after scratch (Supp. Info. Fig.

S5B). We studied the participation of extracellular ATP in

cell permeabilization after scratch at the peripheral region of

the scratch injury in astrocyte cultures. We used a battery of

blockers: suramin (a non-specific blocker of P2 receptors),

oATP (a general blocker of P2X receptors), BBG (a more

selective P2X4–P2X7 receptor blocker), and apyrase VI–VII to

degrade the extracellular ATP. All blockers were applied with

the experimental condition.

Treatment with 200 lM suramin or 10 U/mL apyrase

VI–VII completely prevented the Etd uptake induced by

Trauma and TK1 (Fig. 3D). Indeed, cells treated with sura-

min or apyrase showed an Etd uptake level similar to that of

cells treated with La31. Instead, 100 lM oATP or 10 lM

BBG did not reduce Etd uptake (Fig. 3D), suggesting that a

P2Y-dependent pathway was involved in the activation of

Cx43 HCs. Then, to evaluate the existence of an ATP gradi-

ent concentration effect, we performed a concentration–

response curve with ATP-g-S, a slowly hydrolysable general

P2 receptor agonist. In cultures incubated with increasing

concentrations of ATP-g-S (10 qM to 10 lM) for 1 h, a

proportional increase in Etd uptake occurred only in Control

and Trauma conditions (Fig. 3E). In cells treated with K1 or

TK1, the maximal Etd uptake was already attained at 10

qM ATP-g-S (Fig. 3E). Finally, at 500 lM, there was a gen-

eral decrease in Etd uptake to �30% of the previous value

(Fig. 3E). These data support a differential effect of extracel-

lular ATP on Etd uptake: at low levels (10 qM), the maxi-

mal response occurs only in astrocytes previously exposed to

high [K1]e.

To compare the induction of HC activity after ATP and

elevated [K1]e, with the maximal connexin HC activity feasi-

ble, we studied Etd uptake in a Ca21/Mg21-free aCSF,

named here as 0 Ca21e condition. Divalent extracellular cati-

ons have an inhibitory effect on connexin HC activity, and

their withdrawal leads to connexin HC activation (Stout and

Charles, 2003). In 0 Ca21e, Etd uptake increased up to 4.5-

fold its basal value. Comparatively, iso-osmolar 60 mM [K1]e

or 10 lM ATP-g-S reached �69% and 58%, respectively, of

maximal connexin HC activity (Fig. 3F). Moreover, iso-

osmolar 60 mM [K1]e plus 10 lM ATP-g-S, which repre-

sented the combination of ATP and elevated [K1]e, resulted

in a greater effect nearly reaching maximal connexin HC

activity (Fig. 3F).

To exclude the participation of GJCs in ATP blocking

experiments, we studied whether suramin, oATP, BBG, and

apyrase VI–VII modify dye coupling at 1 h of application.

Except for a decrease in the number of coupled cells by

oATP, none of other blockers significantly changed the inter-

cellular transfer of LY (Supp. Info. Fig. S6A,B).

Secondary Damage Spread is Related to theIncrease in Cx43 HC ActivityThe final step was to study the biological consequence of the

scratch increase in Cx43 HC activity. Because an increase in

connexin HC activity can mediate apoptosis spread (Decrock

et al., 2009), we investigated apoptosis in astrocyte mono-

layers at 24 h after scratch. To this purpose, we evaluated the

presence of annexin V, a molecular marker of apoptosis

(green, Fig. 4A), and labeling with propidium iodide ((PI)

red, Fig. 4A), as an indication of necrosis. The percentage of

apoptotic cells was calculated as the number of annexin V

positive cells minus those co-labeled with PI. We used the

same previous experimental conditions, and five scratches

plus hyper-osmolar 60 mM [K1]e (5TK1) was added to test

1192 Volume 63, No. 7

FIGURE 3: The increase in [K1]e and extracellular ATP mediate the Etd uptake increment after scratch. A: Astrocyte-enriched cultures wereexposed to rapid changes in the extracellular medium composition. 60 mM [K1]e in hyper-osmolar condition (Hi-Osm), was compared with60 mM [K1]e that replaced [Na1]e (iso-Osm); 60 mM sucrose (Hi-Osm), and the combination of these last two conditions. Both changes in osmo-larity and [K1]e increased Etd uptake, compared with their corresponding basal. However, the greatest increase was reached when replacingextracellular K1 with Na1; thus, maintaining an iso-osmolar condition. Each plotted value represents the mean 6 SEM of five independentexperiments (* or #= P < 0.05; ** or ##= P < 0.01; ***= P < 0.001). B: Time-lapse representative measurement of Etd uptake after acute changesof extracellular medium. 60 mM [K1]e iso-Osm produced the greater increase in rate of uptake. C: Concentration response curve between[K1]e added and Etd uptake. There was a positive correlation between [K1]e (Hi-Osm) and Etd uptake. Fluorescence units were normalizedwith respect to the basal value. D: The increase in Etd uptake under the TK1 condition was prevented by 200 mM suramin, a P2 receptorblocker. Its effect was comparable with that of 200 lM La31. Interestingly, 100 lM oATP a general P2X receptors blocker, or BBG, a P2X4 andP2X7 blocker, did not prevent the induction of Etd uptake by TK1. Moreover, inclusion of 10 U/mL apyrase VI and VII (an ATPase) in the extrac-ellular medium prevented the increase in Etd uptake. Each plotted value represents the mean 6 SEM of five independent experiments (*=P < 0.05; **= P < 0.01, n.s., not significant). E: Concentration response curves of ATP-c-S. From 10 qM to 10 lM, there was a linear relationbetween ATP-c-S dose (logarithmic scale) and Etd uptake, for conditions not exposed to high [K1]e: Control and Trauma. In contrast, there wasa plateau curve in these same concentrations of ATP-c-S for conditions with high [K1]e exposition: K1 and TK1. Notably, at 500 lM occurred areduction of Etd uptake for any condition. Each point represents the mean 6 SEM of five independent experiments. F: Simultaneous high K1

e

and extracellular ATP maximize connexin HC activity. Astrocyte enriched cultures were exposed to 0 mM Ca21e, to obtain the maximal open-

ing of connexin HCs. The greater Etd uptake observed after the increment of K1e or extracellular ATP were graphed. Compared with 0 mM

Ca21e, 10 lM ATP-c-S, or 60 mM K1

e (iso-Osm) obtained a lesser Etd uptake. Indeed 60 mM K1e (iso-Osm), only reached ~70% of maximal Etd

uptake. But, when astrocytes are exposed to 10 lM ATP-c-S for 1 h, they reached maximal Etd uptake in the presence of 60 mM K1e (iso-

Osm). Each plotted value represents the mean 6 SEM of five independent experiments (*= P < 0.05; **= P < 0.01; ***= P < 0.001; n.s., not signif-icant difference).

FIGURE 4: Gap26 prevents Cx43 HC opening and apoptosis induced by scratch. Cells were exposed to Control, K1, Trauma, and TK1

conditions, but five scratches plus 60 mM K1e were applied to evaluate the effect of a stronger trauma in vitro. Staurosporine (1 lM St)

was used as positive control. A: Immunofluorescence of cells co-labeled with annexin V Alexa-488 and PI. Nuclei were visualized withHoechst staining. Bar 5 50 lm. B: Graphs showing the percentage of apoptotic cells. Ten fields were quantified per condition. Total pre-vention of scratch-induced apoptosis was observed using the Cx43 HC blocker Gap26, including for the TK1 and 5TK1 conditions. Eachplotted value represents the mean 6 SEM of three independent experiments (*= P < 0.05; **= P < 0.01; ***= P < 0.001). C: Western blotanalysis of cleaved caspase 3. a-tubulin was used as loading control. D, E: ATP and elevated [K1]e induced apoptosis of astrocytes. Toevaluate apoptosis dependent on connexin HC activity, we studied the participation of ATP and elevated [K1]e. Astrocytes wereexposed to 10 lM ATP-c-S or 10 lM ATP-c-S plus 60 mM [K1]e added (Hi-Osm). D: Immunofluorescence of cells co-labeled with annexinV Alexa-488 and PI at 24 h after the experimental conditions. Nuclei were stained with Hoechst, bar 5 50 lm. E: Graphs showing thepercentage of apoptotic cells. Ten fields per condition were quantified. ATP and elevated [K1]e induced apoptosis of astrocytes after24 h. F: ELISA analyses of TNF-a in extracellular medium at 24 or 48 h of scratch and related conditions. Endotoxin shock in adult ratperformed by 500 lg/mL LPS intraperitoneal (ip) injection, and in vitro 1 lg/mL LPS in cultured medium were used as positive controlsfor TNF-a release. Each plotted value represents the mean 6 SEM of three independent experiments. TNF-a levels were not affected bytrauma in vitro and remained low compared with inflammatory levels evocated by LPS (in vivo or in vitro). G: NO2

2 levels (Griessmethod) were measured at 24, 48, 72, and 96 h post scratch and relate conditions. Each plotted value represents the mean 6 SEM offive independent experiments. Nitrite levels were normalized by the Control condition at 24 h. Only five scratches plus 60 mM [K1]eincreased the nitrite level at 48 h (*= P < 0.05).

1194 Volume 63, No. 7

a stronger trauma in vitro. Stauroporine (1 lM) was used as

a positive control of apoptosis (Yue et al., 1998). At 24 h, the

density of apoptotic cells was heterogeneous with density of

death cell clusters similar in all regions (data not show).

However, we observed in all regions counted together an

increased percentage of apoptotic cells at 24 h after Trauma,

TK1, and 5TK1 (Fig. 4A,B). Notably, 5TK1 induced the

highest percentage of apoptotic cells (�18%) compared with

other traumatic conditions (�11%, Fig. 4B). Western blot

analysis for cleaved caspase 3 confirmed apoptosis present in

Trauma and related conditions (Fig. 4C). Then, to evaluate

whether the connexin HC activity was responsible for the

increased apoptosis after scratch, we used Gap26 peptide in

order to prevent the increase in Cx43 HC activity. As illus-

trated in Fig. 4A, and quantified in Fig. 4B, Gap26 peptide

completely prevented the apoptosis induced by the scratch.

Finally, if ATP and elevated [K1]e were upstream of the

increased connexin HC activity after the scratch, they were

probably capable of inducing apoptosis in astrocytes. We used

10 lM ATP-g-S, which produced the greatest connexin HC

activity, in the curve dose response, and hyper-osmolar

60 mM [K1]e to imitate a trauma in vivo. We found 14%

apoptotic cells with 10 lM ATP-g-S, and 15% with ATP-g-

S plus elevated [K1]e (Fig. 4D,E). This outcome was similar

to the percentage of apoptotic cells observed in astrocytes

exposed to the scratch.

The In Vitro Trauma Model Used Produces aDiscrete Inflammation ReactionInflammation is a major source of secondary damage after

trauma (Helmy et al., 2011b), and interacts with connexin

HC activity (Retamal et al., 2007). To quantify inflamma-

tion, we studied extracellular TNF-a and nitrite (as subrogate

for nitric oxide) levels. ELISA analyses of extracellular

medium revealed similar levels of TNF-a at time zero and

after 24 or 48 h of scratch (Fig. 4F). However, there was a

discrete elevation of nitrite levels at 48 h after trauma in vitroreinforced with five scratches (Fig. 4G). Finally, to understand

the potential role of inflammation in scratch-induced

increased Cx43 HC activity, we evaluated Etd uptake after

treatment with inflammatory cytokines. Because time is an

important factor in the evolution of inflammation (Helmy

et al., 2011a), and previous reports from our laboratory

showing that treatment with inflammatory cytokines for 24 h

increases Cx43 HC activity in astrocytes (Retamal et al.,

2007), we assessed Etd uptake under Control, Trauma, and

TK1 conditions after 1 h and 24 h of co-treatment with the

mixture (MIX) of 10 ng/mL TNF-a and 10 ng/mL IL-1b.

An increment in Etd uptake was found only in Control cul-

tures treated with MIX for 1 and 24 h (Supp. Info. Fig. S7).

Treatment with inflammatory cytokines for 1 or 24 h failed

to induce a statistically significant increase in dye uptake by

astrocytes compared with cells treated with Trauma or TK1

alone (Supp. Info. Fig. S7).

Discussion

We demonstrated that Cx43 HCs were responsible for the

increase in membrane permeability observed in astrocytes 1 h

after trauma in vitro. This increase in HC activity was associ-

ated with secondary damage spread, which resulted in apo-

ptosis. The mechanisms that appeared to be involved in HC

activation were the acute increase in [K1]e and signaling by

extracellular ATP.

Several pieces of evidence indicate that increased cell

membrane permeability after scratch was due to activation of

Cx43 HCs. First, we found clusters of Etd uptake at periph-

eral ROIs after scratch that were most intense after scratch

plus hyper-osmolar 60 mM [K1]e. Second, increased Etd

uptake was blocked by La31, which is a connexin HC blocker

that does not inhibit Panx1 channel (Pelegrin and Surprenant,

2006), and was not affected by RuR, excluding CALMH1

participation (Taruno et al., 2013). Third, in line with the

previous result, Gap26 but not 10panx1 prevented the

increase of Etd uptake after Trauma and TK1. Fourth, unlike

WT, Cx43 KO astrocytes did not reproduce the increase in

Etd uptake after Trauma and TK1. Fifth, the functional state

of GJCs evaluated through a dye coupling technique

remained unchanged after trauma or TK1, and was not

affected by La31 or Gap26 peptide, indicating that GJCs did

not have a large contribution to the studied phenomenon.

Our results extend the work of Frantseva et al. (2002)

that demonstrated the participation of connexins in trauma-

induced cell death, using a pharmacologic and transgenic

approach in organotypic hippocampal slices. However, only

participation of GJCs was evaluated. Here, we demonstrated

the participation of Cx43 HCs 1 h after scratch-trauma invitro. Our results are in agreement with those obtained by

O’Carroll et al. (2008), who also suggested Cx43 HCs partic-

ipation in initial damage spread, after spinal cord trauma.

However, we cannot discard a relevant participation of GJCs

mediated communication at later stages post trauma. In fact,

an in vivo trauma model reported an increased immunoreac-

tivity for Cx43 in hippocampal astrocytes ipsilateral to the

injured hemisphere, at 72 h after trauma (Ohsumi et al.,

2010). Similarly, an increased punctate staining, typical of

Cx43 gap junction plaques, was reported at the injury site in

a stab wound brain trauma model, from days 3 to 15 post

trauma (Theodoric et al., 2012).

To evaluate the mechanism involved in the increment of

Cx43 HC activity, we studied conditions that resembled in

vivo trauma. During trauma, a variable destruction of cells

occurs due to primary damage. The ruptured cells dump their

Rovegno et al.: Astroglial HCs and Damage Propagation in Trauma

July 2015 1195

intracellular content into the extracellular medium. In fact,

the osmolarity of the extracellular solution in the damaged

brain tissue is higher than 400 mOsm (Kawamata et al.,

2007) and there is an acute and transitory elevation of [K1]e

close to 55 mM (Nilsson et al., 1993). Here, we demon-

strated that an increment of [K1]e to levels similar to those

found during brain trauma increased Etd uptake, and clearly

potentiated the in vitro trauma effect, suggesting that it could

enhance membrane permeability of astrocytes located near the

scratch region. Even more, a rapid increment in [K1]e and

osmolarity produced a rapid increase in Etd uptake. This was

most pronounced in iso-osmolar aCSF, accomplished by

replacing [Na1]e with [K1]e. Similarly, opening of Cx50

HCs induced by replacement of extracellular Na1 with K1

has been demonstrated (Srinivas et al., 2006). However, it is

still unknown whether HCs formed by different connexin

types are activated by high [K1]e.

In our in vitro study the increase in [K1]e induced by

the scratch was minimal, given that the extracellular volume

is several orders of magnitude larger than the volume of cells

destroyed by the scratch. This could explain the small but sig-

nificant ATP extracellular concentration and the values of

[K1]e found after injury. However, in vivo the extracellular

space is limited and the increment in [K1]e is similar to that

applied in the experiments described here.

Thus, on an in vivo setting, increment of Cx43 HC

activity could be induced to a large extent by the increase in

[K1]e. However, other factors could also contribute, because

increased [K1]e alone did not increase Etd uptake in the

same extent to that induced by the scratch. Accordingly, we

found that extracellular ATP and P2 contributed to HC acti-

vation as previously described in tanicytes and astrocytes

(Orellana et al., 2012; S�aez et al., 2013). This result is con-

sistent with previous ATP-trauma findings. For example, there

is an ATP increment after in vitro trauma (Ahmed et al.,

2000; Neary et al., 2005), ATP appears to open pannexin

channels and connexin HC in spinal cord astrocytes (Garr�e

et al., 2010), and in a recent study, inhibition of connexin

with carbenoxolone prevented a purinergic dependent inflam-

matory response in a TBI model (Roth et al., 2014). Further-

more, given that ATP released by trauma in vitro rapidly

increases and then declines over 1 h (Ahmed et al., 2000;

Neary et al., 2005), there is a temporal window to form gra-

dients. We showed the participation of ATP for increasing

connexin HC activity after the scratch, based on two inde-

pendent approaches. First, we observed that ATP receptor

blockers with P2 blockade or by extracellular ATPase action

blunted connexin HC activity after the scratch. From our

experiments, it is not possible to ascertain the exact identity

of the P2 receptor involved, but the absence of blocking with

oATP and BBG suggest that the P2Y pathway is especially

involved. Second, a concentration–response curve with a

more stable analogue of ATP (ATP-g-S) affected connexin

HC activity only in the absence of previous elevation of

[K1]e. Remarkably, high ATP-g-S caused less increase in Etd

uptake. This suggests the existence of a gradient effect: only

with small increases in ATP levels, like those probably

reached at peripheral regions, ATP increased connexin HC

activity. Also, this argument favors the participation of P2Y

pathway, because P2Ys are more sensitive to ATP than most

P2X receptors, and signals at low concentrations of purinergic

analogues (Fischer and Kr€ugel, 2007; Fischer et al., 2009). In

order to consider the contribution of GJCs, we showed that

astrocytes maintained their intercellular coupling at 1 h after

scratch. Permeable GJCs enable a continued transfer of intra-

cellular metabolites, including ATP, when healthy cells are

coupled with the injured cells. In essence, forming a gradient

of metabolites in the extracellular milieu from the scratch

area to the periphery. Finally, the presence of this ATP gradi-

ent could explain why the connexin HC activity increment

was more evident at peripheral ROIs.

If the [K1]e and ATP effects are complementary, this poses

an interesting question because a priory, the role of [K1]e pro-

posed for opening of connexin HC is by a rapid effect of K1 on

the HCs, and probably affects the Ca21 binding site (Srinivas

et al., 2006). We assessed whether this last effect was responsible

for the modulation by [K1]e of the response after 1 h. ATP and

[K1]e could be complementary since at low levels of ATP-g-S,

the Control and K1 conditions reached the same level of Etd

uptake as Trauma and TK1 without ATP-g-S. In addition,

ATP-g-S and elevated [K1]e were complementary in augmenting

the level of HC activity, comparable to that obtained with a low

Ca21/Mg21 condition. Probably, in trauma there is a coexistence

of two ways to increase HC activity for the ATP and K1, with a

complementary effect. However, these data must be interpreted

with caution because the maximal HC activity obtained with low

Ca21/Mg21 condition is not a physiologic state. In contrast, the

action of K1 on Cx43 HCs over time and its interaction with

ATP signaling remains to be determined.

The increment of connexin HC activity induced by

scratch might be expected to be related with cell death, and

manipulation of HCs could avoid cell damage (Contreras

et al., 2002; Decrock et al., 2009). To test this point, we ana-

lyzed the percentage of apoptotic cells after 24 h of the

scratch and after Cx43 HC activity was blocked by Gap26

peptide, thus we observed a total prevention of apoptosis.

Although a previous work have showed direct spatial correla-

tion between connexin HC activity and cell death, using an

in vitro model of injury induced by electroporation of cyto-

chrome C (Decrock et al., 2009), we found random distribu-

tion of cell death clusters with regard to distance to the

scratch. Without ruling out other possibilities, this apparent

1196 Volume 63, No. 7

controversy might be explained by differences in cell hetero-

geneity; a cell line (rat C6 glioma cell line) is likely to be

more homogenously susceptible to death than primary cul-

tures of cortical astrocytes.

Notably, extracellular ATP and elevated [K1]e induced

apoptosis at similar levels than in the injury scratch wounding

model used in the present work. These findings are consistent

with a common pathway regarding secondary injury propaga-

tion, namely that extracellular elevation of K1 and ATP

enhance the scratch-associated induction of Cx43 HC activity

resulting in cellular apoptosis. Activation of connexin HC

carries a potential danger to cells, a fact known for other con-

nexin types, and also for Cx43 (Decrock et al., 2009).

Inflammation is a well-established secondary response

associated with acute brain injury. In brain trauma, infiltrated

and local cells (i.e., microglia and astrocytes) trigger an

inflammatory cascade involving intercellular communication

mediated by cell–cell interactions and pro-inflammatory mol-

ecules (Bennett et al., 2012; Finnie, 2013; Orellana et al.,

2009). Inflammatory changes appear with a delay of several

hours, and peaks from 12 h to 2 days after trauma (Helmy

et al., 2011a). In the trauma model used here, only a discrete

inflammatory response was detected as evidenced by elevation

of nitrite levels at 48 h after the scratch injury, but no signifi-

cant increment in TNF-a was found. These findings contrast

with those of Lau and Yu (Lau and Yu, 2001), who described

a significant increment of inflammatory cytokines after

scratching astrocyte monolayers. However, the two models

have important differences in the traumatized area. Whereas

�40% of the culture surface was scratched in the study by

Lau and Yau, it was less than �1% in our model. That could

explain the differences in the inflammatory outcome. In fact,

we considered that the use of a model with almost no inflam-

mation was an advantage. Inflammation induces drastic

changes on connexin-based channels functions. Particularly,

24 h of treatment with TNF-a and IL-1b increase the Cx43

HC activity and decrease cell–cell coupling in astrocytes

(Morita et al., 2007; Retamal et al., 2007). Here, we demon-

strated the existence of an early modification of connexin HC

function that explains the spread of secondary damage. Fur-

thermore, in our study stimulation with TNF-a and IL-1b

did not increase connexin HC activity above the scratch-

induced effect at 1 or 24 h of treatment.

Although these results were obtained from astrocyte-

enriched cultures, they provide new insight about the early

mechanisms that take place during the cellular spread of sec-

ondary damage. As illustrated in Fig. 5, we highlighted how

intrinsic components of trauma, namely elevated [K1]e and

ATP, which are part of the primary damage of cells, can acti-

vate a connexin HC-dependent pathway by means of a direct

action of [K1]e and ATP via P2 receptors. Accordingly, it has

been shown that Cx43 HCs can contribute to the propaga-

tion of necrosis in models of in vitro ischemia (Contreras

et al., 2002; Orellana et al., 2010) as well as propagation of

apoptosis induced by cytochrome C (Decrock et al., 2009).

To our knowledge, this is the first demonstration on extracel-

lular ATP-mediated activation of connexin HC leading to

post trauma cell death propagation. Therefore, inhibition of

Cx43 HCs could reduce the spread of secondary cell damage

and thus could open a glia-centered approach for therapeutic

applications in brain trauma.

Acknowledgment

Grant sponsor: Direcci�on de Investigaci�on de la Facultad de

Medicina-PUC; Grant number: PMD-09/09; Grant sponsor:

CONICYT; Grant number: AT-24100202; Grant sponsor:

FONDECYT; Grant number: 1131025; Grant sponsor:

Anillo; Grant number: ACT-71; Grant sponsor: FONDEF;

Grant number: D07I1086; Grant sponsor: Instituto Milenio,

Centro Interdisciplinario de Neurociencia de Valpara�ıso;

Grant number: P09-022-F; Grant sponsor: Heart & Stroke

Foundation of BC & Yukon.

Authors thank Ms. Teresa Vergara, Ms. Paola Fern�andez,

and Ms. Gigliola Ramirez for their technical support.

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FIGURE S1: characterization of glial cell cultures. A: Cell identity of cultures was evaluated by immunofluorescence. Cells were co-labeled for GFAP (green cells, astrocytes) and isolectin B4 (red cells, microglia). Nuclei were stained with Hoechst (blue). Panels (a) & (b) are low power photomicrographs

showing the injury distribution and nuclei labeling (a) and overlay of nuclei and GFAP labeling (b). Panels (e) & (f) are high magnifications of the center and periphery of the previous images, and (d) is the bright

field of the image in (e). (c) Positive control of isolectin B4 marker in a primary rat microglia culture, bar = 50 µm. There is a predominance of astrocyte in the cultures and (f) the few microglia are highlighted with

white arrows. B: Graphs showing the average of cell identity expressed as percentage. Ten fields were

quantified by experiment. Each plotted value represents the mean ± SEM; n = 5.

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FIGURE S2: In vitro trauma model. Trauma was performed by a single scratch along the middle of a 35 mm culture dish to rat astrocyte-enriched monolayer, using a 25 G needle. A: Bright field of trauma by scratch that produced an ~60 µm wide injury, bar = 50 µm. B: Three experimental regions were defined

according to their increased distances from the injury site as indicated.

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FIGURE S3: Trauma and TK+ transiently increase connexin HC activity in peripheral regions of interest (ROI) at 1 h. A: Etd uptake (snap-shots) of peripheral ROIs under different conditions at different times,

and their respective bright field, bar = 50 µm. B: Time course of Etd uptake at peripheral ROIs. The

increased Etd uptake profile across conditions was maximal at 1 h post injury. Each value represents the mean ± SEM of the difference between Etd uptake inductions minus the Etd uptake after 200 µM La3+. Value

represents the mean ± SEM of n = 3 independent experiments (**= p<0.01).

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FIGURE S4: Cx43 HCs mediate the Etd uptake increment after TK+. A: Representative photomicrographs of Etd uptake. Gap26 peptide prevented the TK+-induced Etd uptake, bar = 50 µm. B: Time-lapse

measurement of Etd uptake of astrocyte cultures under TK+ without and with Gap26 peptide. The TK+-

induced Etd uptake was reduced by 150 µM Gap26, and was insensitive to 200 µM La3+. Under Control condition the reduction of Etd uptake after 200 µM La3+ was similar to the TK+ condition treated with Gap26 peptide. C: Photomicrographs of cultures exposed to 20 µM Ruthenium Red, a blocker for CALMH1 ion

channels. D: Average dye uptake under Control and TK+ condition, Ruthenium Red decreased Etd uptake to similar levels for both conditions (~19%). This finding indicates that Ruthenium Red did not change the Etd uptake increased after TK+ condition. Each plotted value represents the mean ± SEM of 5 independent

experiments (n.s., not significant difference).

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FIGURE S5: Extracellular ATP increases after trauma in vitro. To study the increase on ATP and [K+]e in response to the in vitro trauma model, we exposed astrocyte monolayers to one or five scratches, and measured ATP & [K+]e on samples of extracellular medium, after 1 h of incubation. [K+]e was measured

using a potassium ion selective electrode, and ATP by bioluminescent assay using luceferin – luciferase reaction. A: There was not a significant increment of [K+]e after Trauma or 5T. B: Extracellular ATP

concentration expressed in logarithmic scale. Compared with the Control, Trauma and 5T produced a small but statically significant increase of extracellular ATP. Values represent the mean ± SEM of 3 independent

experiments (*= p<0.05).

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FIGURE S6: HC blockers do not affect the functional state of GJCs. The possible effect of blockers used in experiments on gap junction communication was tested using dye coupling in monolayers of rat astrocytes. Panels A and B correspond to the index and dye coupling incidence evaluated 1 h after the application of the

indicated compound (200 µM La3+, 150 µg/ml Gap26, 50 µM β-GA, 200 µM suramin, 10 µM BBG, and 100 µM oATP). With the exception of β-GA and a partial effect of oATP, others blockers did not modify astrocytes

coupling. Values represent the mean ± SEM of 3 independent experiments (***= p<0.001).

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FIGURE S7: Inflammation did not modify connexin HC activation after trauma-scratch. The effect of inflammation on scratch-induced changes was tested by treating the cultures with 10 ng/ml TNF-α plus IL-1β. Etd uptake was evaluated at 1 and 24 h later. Compared with the unstimulated conditions, only control

astrocytes showed an increased Etd uptake after 1 or 24 h of cytokines incubation. Data is presented as normalized values of fluorescence. Each bar represents the mean ± SEM of 3 independent experiments (*=

p<0.05). 97x54mm (600 x 600 DPI)

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