connexin43 antisense oligodeoxynucleotide treatment down-regulates the inflammatory response in an...

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Journal of Clinical Neuroscience 15 (2008) 1253–1263

Laboratory Study

Connexin43 antisense oligodeoxynucleotide treatmentdown-regulates the inflammatory response in an in vitro interphase

organotypic culture model of optic nerve ischaemia

Helen V. Danesh-Meyer *, Rex Huang, Louise F.B. Nicholson, Colin R. Green

Department of Ophthalmology, Private Bag 92019, Auckland, 1020, University of Auckland, New Zealand

Received 24 August 2008; accepted 24 August 2008

Abstract

Using a model of optic nerve ischaemia, this study investigated oxygen-glucose deprivation (OGD) on isolated rat optic nerve seg-ments cultured in vitro. Thereafter, the effect of antisense oligodeoxynucleotides (ASODN) specific to the gap junction protein connex-in43 (Cx43) was evaluated in this same model. Following exposure to OGD for 2 hours, optic nerves were maintained in interphaseorganotypic culture with and without exposure to Cx43 ASODN. Optic nerves were sectioned at 2 hours, 6 hours, and at days 1, 2,3 and 6 following culture. Cell death was quantified using propidium iodide (PI) staining and specific markers for Cx43, capillaries(von Willebrand factor), astrocytes (glial fibrillary acidic protein), microglia and endothelial cells (isolectin B4) were used to evaluatethese parameters in conjunction with digital light and confocal microscopy. In this model, up-regulation of Cx43 was seen at 2 hoursfollowing exposure of the optic nerve to OGD and peaked at day 3. Cx43 ASODN treatment dampened this up-regulation. Additionally,more PI labeled cells were found in the centre of control optic nerve segments than in treated nerves (p < 0.01). Controls also showedevidence of capillary breakdown and increased numbers of astrocytes and activated microglia compared to Cx43 ASODN treated nerves(p < 0.05). Thus, the application of Cx43 ASODN to post-ischaemic optic nerve segments significantly reduced the up-regulation of Cx43and, subsequently, the spread of injury and a resultant inflammatory response. Cx43 up-regulation may play an important role in opticnerve injury, offering a potential avenue for treatment in optic neuropathy.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Connexins; Optic nerve; Organotypic culture; Central nervous system inflammation

1. Introduction

After an ischaemic insult to the central nervous system(CNS), a complex cascade of events occurs that ultimatelyleads to cell death and neurological damage. Intercellularcommunication via gap junctions may play a role in ischae-mia-induced cell death and may also contribute to thespread of damage to areas outside the area affected bythe initial insult, a process known as the gap junction-med-iated bystander effect.1 Gap junctions serve as intercellular

0967-5868/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jocn.2008.08.002

* Corresponding author. Tel.: +64 21 229 1840.E-mail address: [email protected] (H.V. Danesh-

Meyer).

conduits that allow for the direct transfer of smallmolecular weight molecules (up to about 1 kDa) includingions involved in cellular excitability, metabolic precursors,and secondary messengers such as calcium and gluta-mate.2–6

Gap junctions are formed by the joining of two hemi-channels (connexons) from adjoining cells, which are inturn comprised of six transmembrane peptides, termedconnexins.5–7 In the adult brain the predominant connexinis connexin43 (Cx43), which is abundant in astrocytes andalso expressed in the leptomeninges, endothelial cells, andependyma.1 Gap junctions can open under both physiolog-ical and pathological conditions, and opening can be eitherfunctional or deleterious, depending on the situation. Sev-eral publications report that up-regulation of Cx43 can

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1254 H.V. Danesh-Meyer et al. / Journal of Clinical Neuroscience 15 (2008) 1253–1263

occur with mechanical loading of the blood vessel wall andin vitro cell stress.8,9

A specific connexin can be inhibited by means of anti-sense oligodeoxynucleotides (ASODN), short chain nucle-otides that can block expression of a target gene bybinding to and preventing translation of mRNA, thus tem-porarily reducing target protein production. Application ofCx43-specific ASODN results in a temporary knockdownof Cx43 production and a decrease in the formation ofnew Cx43 gap junctions.10 Unmodified ASODNs have ashort half-life of about 20 min after which they are de-graded by nucleases within the cell.11 Incorporation of aPluronic F-127 gel delivery system (Sigma-Aldrich, StLouis, MO, USA) aids penetration of ASODN into cellsand act as a reservoir to provide sustained release ofASODN, thus ameliorating the effect of intracellular nucle-ase breakdown.12 Knockdown of Cx43 expression can beachieved for 24 hours to 48 hours using this gel deliverysystem.13

We investigated whether Cx43 is up-regulated in theoptic nerve under ischaemic conditions and, if so, whetherthis up-regulation would lead to increased tissue damage.In this investigation organotypic culture methods for theculture of excised segments of rat optic nerve were adaptedand used to study the effect of Cx43 mRNA-specificASODN after 2 hours of anoxia and hypoglycaemia.

2. Methods

2.1. Animals

Wistar rats aged from 21 days to 25 days post-natal wereused. Animals were obtained from the Vernon Jansen Unit(VJU), University of Auckland. All animal procedures inthis study were approved by the Animal Ethics Committeeat the University of Auckland and were in accordance withthe Association for Research in Vision and Ophthalmology(ARVO) Statement for the Use of Animals in Ophthalmicand Vision Research. Rats were housed under standardconditions and fed food and water ad libitum. Animalswere euthanatized by carbon dioxide (CO2) inhalation,after which tissues were harvested and prepared for appro-priate assays.

2.2. Dose response analysis of efficacy of ASODN

Based upon previous studies conducted in our labora-tory, three concentrations of Cx43 specific ASODN weretrialled in the present study, 2.5 lM, 5 lM, and 10 lM.The optimal concentration was determined using twoparameters: (i) percentage of swelling ([area after culture– original area]/original area � 100); and (ii) the numberof dead cells identified using propidium iodide (PI) nuclearstaining. We assessed these parameters in a simple cut-endexcised optic nerve model in which damage spreads fromthe cut ends.

2.3. Dissection and preparation of the optic nerve

Optic nerves were harvested using an intracranial ap-proach. The skull was opened in a midsagittal orientationwith the cerebral tissue caudal to the cerebellum carefullyexcised and discarded. Incisions were made below theolfactory lobes to reveal the intracranial region of the opticnerve. We obtained 3 mm to 5 mm of optic nerve thatspans the anterior cranial fossa from the chiasm to the op-tic canal. After dissection, the optic nerves were transferredto a sterile filter paper wetted with Neurobasal mediumwith B27 supplement, D-glucose, L-glutamine and antibi-otics (Gibco; New York, NY, USA). The two ends of theoptic nerve were cut with a surgical knife to produce cleanedges. The optic nerve was then placed on Millicell-CMculture plate inserts (Millipore, Bedford, MA, USA) andsubsequently placed into a six-well plate containing 1 mLof the medium per well. Three experimental groups wereset up in this fashion: (1) nerves treated with 7 lL of2.5 lM, 5 lM, or 10 lM Cx43 specific ASODN in 30%w/v Pluronic gel (#P2443, Sigma-Aldrich); (2) nerves withthe Pluronic gel only; and (3) control optic nerves withoutany treatment. This resulted in the application of doses ingroup 1 equal to about 0.2 lg, 0.35 lg, and 0.7 lgrespectively.

2.4. In vitro ischaemic optic neuropathy model

ASODN treatment parameters were assessed using asimple cut-end excised optic nerve model in which damagespreads from the cut ends. For the main portion of thestudy, we introduced optic nerve ischaemia. An in vitro

ischaemic optic neuropathy model, based on previous tech-niques established by Sundstrom et al.,14 was modified inorder to evaluate the effect of ischaemia on the supportingglial tissue (astrocytes and microglia) and vasculature with-in the isolated optic nerve segments. An ischaemic solutionwas prepared by placing 10 mL of Neurobasal medium in afalcon tube without glucose and glutamine. The solutionwas bubbled with a 95% nitrogen (N2) and 5% CO2 gas mix-ture for 30 min to remove all the oxygen. The dissected op-tic nerves were transferred to the oxygen-glucose deprived(OGD) solution and the tubes closed and sealed with plasticfilm. These optic nerves were then incubated in the ischae-mic solution for 2 hours at 37 �C and 5% CO2 and wereplaced subsequently into normal organotypic culturing con-ditions for various lengths of time. Control nerves wereplaced directly into organotypic culture (see below).

2.5. Organotypic interphase cultures

After incubation in OGD solutions, the optic nerveswere placed onto an air-liquid interphase organotypic cul-ture. Organotypic cultures, initially introduced by Gahwil-er in 1981, allow CNS tissue to maintain organisationalfeatures of the host tissue such as neuronal connectivity,relatively well-preserved cellular stoichiometry, and com-

Fig. 2. Dose response curves in a cut end model to establish the optimumantisense concentration used for subsequent experiments. (A) Thepercentage of nerve swelling plotted against dose at two different timesafter placing into culture (6 h, blue line; 24 h, green line). At both times,the swelling is minimum at the 10 lM concentration. (B) The number ofdead cells per field of view with increasing concentration of antisenseoligodeoxynucleotides (ASODN) used at 24 hours after placing intoculture. Front refers to the eye end of the optic nerves (green line) with themid being the central region of the nerve (blue line). For both areas there isreducing cell death with increasing dose, although the main benefit isachieved within the 5 lM concentration. For all further experiments, a10 lM concentration of ASODN was used in the ischaemic optic nervemodel.

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plex glial-neuronal interactions.14 We placed 24 optic nervesections onto culture inserts containing a semi-porousmembrane (Millicell-CM). The inserts were in a six-wellplate, each well containing 1 mL of Neurobasal mediumwith B27 supplement, D-glucose, L-glutamine, and antibi-otics per well. Once placed onto the inserts (up to 3 nervesper insert), the optic nerves were coated with ice cold 30%w/v Pluronic F-127 gel containing antisense (see below), gelonly, or medium. The thermo-reversible gel is a liquid at4 �C but sets quickly as it warms to room temperature(see for example, Qiu et al.10). The organotypic cultureswere then placed into an incubator (5% CO2, 37 �C) forthe required culture period. This organotypic culture set-up is shown in Fig. 1. The semi-permeable insert allows dif-fusion of nutrients from the medium (red arrows) from theunderside of the membrane and constant oxygen supplyfrom above (arrow heads).

For antisense treatment 7 lL of 30% w/v Pluronic F-127gel containing 2.5 lM, 5 lM, or 10 lM ASODN specificfor Cx43 was administered to cover each optic nerve. Theamount is sufficient to cover the whole segment withoutflooding the tissue. The sequence used, 50GTAATT-GCGGCAGGAGGAATTGTTTCTGTC30, is specific forCx43 as confirmed by sequence alignment using the genebank (GenBank) of the National Center for BiotechnologyInformation (NCBI) website and has been shown to beeffective in knocking down Cx43 expression.10,13,17 Forgel only and control groups, the same amount (7 lL) ofPluronic F-127 gel or medium was applied to the opticnerve, respectively. Dose response curves (percentage swell-ing and amount of cell death) were generated (see Resultsand Fig. 2) and subsequently, the main study experimentswere conducted using 10 lM ASODN concentration withoptic nerves (antisense n = 12), gel only (n = 6) or medium(n = 6) cultured to various type points: 2, 6, and 18 hours,and 1, 2, 3, and 6 days.

2.6. Cell death visualisation – propidium iodide staining

After culture, the nerves were rinsed in phosphate-buf-fered saline (PBS) (#BR14, Oxoid, Cambridge, UK), and

Fig. 1. The organotypic culture model used in this study. (A) A top view of thesix-well culture plate, and coated with a droplet of either antisense, gel, or mediis placed into a culture plate well. The culture medium is raised just to the bpermeable membrane insert allows diffusion of nutrients from the medium (re

incubated for 20 min in medium containing 0.01 lg/lL PIto identify necrotic or apoptotic cells.15 The optic nerveswere then washed in PBS for 10 min and fixed in 4% para-

experimental set-up with the optic nerves sitting on a membrane insert in aum. (B) A schematic representation of the components. A membrane insertottom of the optic nerve, forming a meniscus over the nerve. The semi-d arrows) and an oxygen supply from above (arrow heads).


formaldehyde (PFA) for 2 hours, followed by cryoprotec-tion with 30% sucrose in PBS. The nerves were then storedin 15% sucrose in PBS at 4 �C. The optic nerves wereembedded in an optimum cutting temperature (OCT) for-mulation of water-soluble glycols and resins (#4583, Tis-sue-Tek; SakuraFinetek, Torrance, CA, USA), frozen at�20 �C and sectioned longitudinally, parallel to the axisof the axons, into 16 lm thick sections. The sections weremounted onto Histobond slides (#0810001, Marienfeld,Germany).

2.7. Labeling and immunohistochemistry

Microglial cells and blood vessel endothelial cells wereidentified using isolectin B4 (IB4) conjugated to horse rad-ish peroxidase (HRP).16 Sections were washed in PBS andendogenous peroxidase activity blocked with 50% metha-nol, 1% hydrogen peroxide (H2O2) and 49% H2O. Sectionswere incubated overnight with 2.5 lg/mL of IB4 in PBS,washed in PBS (3 � 1 hour), and then incubated with 3,3-diaminobenzidine (DAB) solution for 1.5 min (1% H2O2,10% DAB, 89% PBS buffer). After the color reaction, theslides were washed in PBS and cover slips mounted.

For immunohistochemical labeling of astrocytes, bloodvessels and gap junctions, frozen slides were thawed atroom temperature for 30 min before rinsing in PBS to re-move residual OCT. This was followed by 1 hour blockingwith 0.1% Triton X-100 and 1% normal goat serum (NGS)to facilitate the access of antibodies and to block non-spe-cific binding, respectively. The pre-treated sections werethen incubated with primary antibodies (Table 1) over-night. After washing in PBS for at least 3 hours, secondaryantibodies were applied for 2 hours, followed by washing inPBS (3 � 15 min). The slides were then blotted dry,mounted with Citifluor (Agar Scientific; Stansted, Essex,UK) and sealed with a cover slip and nail varnish. To per-form double labeling, two single labeling procedures werecarried out sequentially, followed by mounting and coverslipping.

Table 1Antibodies used to label astrocytes, blood vessels and gap junctions

Cell type Primary antibody Dilution Provider

Astrocytes Mouse anti-GFAPCy3conjugated

1:1000 Sigma, St LouiUSA

Vessel endothelial cells Rabbit anti-vWF 1:200 Dako, GlostruDenmark

Cx43 Gap junctionprotein

Mouse anti-Cx43 1:200 Dr David BeckUCL

The Cx43 specific gap junction antibody targets the cytoplasmic carboxyl tail ofCollege London.Cx43 = connexin43, Cy3 = cyanine labeled, GFAP = glial fibrillary acidic pro

2.8. Imaging

Sections labeled for IB4 were examined for horseradishperoxidase (HRP) reaction product using a digital lightmicroscope (Leica DMRA, Germany with Nikon camera,Nikon Instruments, Melville, NY, USA), while the glialfibrillary acidic protein (GFAP), von Willebrand factor(vWF) and Cx43 labeled sections were imaged using eithera Leica SP2 confocal laser scanning microscope (CLSM)(Leica Microsystems, Wetzlar, Germany) or TCS-4DCLSM (Leica Microsystems) situated in the BiomedicalImaging Research Unit, University of Auckland. To visu-alize Alexa-488 labeled structures with the SP2 CLSM,an argon ion laser with 488 nm wavelength excitation wasused. The signals were detected through a 500 nm to530 nm band-pass filter with one Airy disk diameter pin-hole. Cyanine (Cy3)-labeled tissue was observed using agreen helium neon laser with 561 nm excitation and a570 nm to 700 nm band-pass filter, also through one Airydisk diameter pinhole. On the TCS-4D microscope both la-bels were visualized using an argon-krypton laser with488 nm and 568 nm excitation, respectively, and detectedusing selected emission filters sets.

3. Results

3.1. Dose response – decreased oedema and cell death

In vitro optic nerve swelling was expressed as the per-centage of swelling from original size to account for anydifference in size of the optic nerves immediately afterischaemia. All images were taken with a stereo-light micro-scope with 1.25 zoom and 8 � final magnification.

The percentage of oedematous area and number of deadcells decreased with increasing ASODN concentration(from 2.5–10 lM) in our cut-end model. At both 6 hoursand 24 hours post-ischaemia there was a clear dose-re-sponse relationship (Student’s t-test analysis) between thepercentage of swelling (p < 0.05) and ASODN concentra-

Secondary antibody Dilution Provider

s, MO, - - -

p, Goat anti-rabbit Alexa488

1:300 Molecular Probes,Invitrogen, Eugene,OR USA

er, Rabbit anti-mouse Cy3 1:200 Jackson,Immuno-Research,West Grove, PA,USA

the Cx43 protein and was kindly provided by Dr David Becker, University

tein, UCL = University College London, vWF = von Willebrand factor.


tion (p = 0.01); in the control nerves, swelling was 16% ofthe initial optic nerve area at 24 hours (viewed from above)compared to 4% in nerves antisense treated at the higher10 lM dose (Fig. 2A).

At 24 hours we observed an increased reduction in celldeath at both the cut edges and towards the centre (mid re-gion) of the optic nerves with increasing ASODN concen-tration (Fig. 2B). For all remaining studies, the opticnerves were, in addition, exposed to 2 hours of ischaemia- a 10 lM antisense concentration was used.

3.2. Cultured tissue morphology

Ischaemic incubation induced slight swelling in the tis-sue. Subsequent culturing in organotypic solution resulted

Fig. 3. Nerve swelling in control and connexin43 (Cx43) specific antisense oligoASODN treated (right) nerves. With oedema the tissue, constrained by the diamlike morphology. The swelling at the ends of the control nerve is greater thanCx43 specific ASODN treated nerves graphed over time. Control nerves swellnerves (blue line). The red asterisks indicate that the differences between cont(p < 0.05).

in additional optic nerve swelling in both control andASODN treated nerves. In both groups, the swelling ap-peared predominantly at the cut ends of the optic nervewithin 24 hours giving the tissue a ‘‘dog bone” like mor-phology (Fig. 3A). (Because the nerve is contained withina sheath, the effect of nerve oedema is to squeeze out theends like toothpaste from a tube, creating the dog bone ef-fect). This is consistent with previous findings in spinal cordcultures in our laboratory.17 Greater expansion of the oede-matous area was observed in nerves following 2 hours ofischaemia compared with cut end damage alone, with thetissue swelling in excess of 25% in controls within 24 hoursafter ischaemia (compared with 16% with cut ends only).

In contrast, Cx43 ASODN treatment reduced this swell-ing by more than 50%. Just over 10% of swelling was noted

deoxynucleotide (ASODN) treated nerves. (A) Images of control (left) andeter of the nerve sheath, is squeezed from the ends to form a ‘‘dog bone”-

for the treated nerve. (B) The percentage of nerve swelling in control andby over 25% within 24 hours (green line) compared to just 10% in treatedrol and treated nerves are statistically significant for all three time points


for the ASODN treated group; a 60% reduction from con-trol (compared with 4% in those with cut ends only, a 75%reduction from control). The difference between ASODNtreated ischaemic nerve segments and controls was signifi-cant at all time points assessed (2, 18, 24 hours, Student’st-test, p < 0.05) (Fig. 3B). These data show that the effectof ischaemia following initial insult (cut ends and ischae-mia) led to an approximate doubling in the amount ofoedema.

3.3. Propidium iodide (PI) staining

Using ImageJ software (open source) cell death in theoptic nerve ischaemia model was quantified at the cut edgeand in the centre of the optic nerve at 2 hour, 6 hour, and24 hour time points. PI staining at the cut edge of the opticnerve showed a statistically significant difference (Student’s

Fig. 4. Staining of dead cells with propidium iodide in control and connexin4nerves. At the cut edges of optic nerves, cell death was greater in the controls agroup control, but by 24 hours (A) control nerves and (C) ASODN treated nervnerve excision. (B) The effect of ischaemia was most evident in the centre of coninduction. The Cx43 ASODN has been protective in treated nerves where theredifference in the centre between control and treated optic nerves remained aft

t-test, p = 0.01) between the control and ASODN treatednerve segments per field of view at 2 hours (225 dead cellscompared with 90) and 6 hours (170 dead cells comparedwith 95), but there was no difference between the twogroups at 24 hours (190 dead cells compared with 150; Stu-dent’s t-Test, p = 0.6) (Fig. 4A,C). At 24 hours, however,this largely reflects the damage incurred during nerve exci-sion (see Fig. 2B showing the dose response curve for deadcells near the ends and in the centre for cut end damageonly).

In the middle region of the optic nerve where cell deathresults from lesion spread or ischaemia itself, a distinct andsignificant difference between the control and treated nervesegments was noted at all time points with significantlylower cell death in the Cx43 ASODN treated optic nerves(20 dead cells per field of view compared with 65 at 2 hours,20 dead cells compared with 95 at 6 hours, and 30 dead

3 (Cx43) specific antisense oligodeoxynucleotides (ASODN) treated optict 2 hours and 6 hours after ischaemia compared with the ASODN treatedes showed similar levels of cell death. However, this reflects damage duringtrol nerves with large numbers of dead cells within 6 hours after ischaemicare few dead cells present in the centre (D) compared to the controls. This

er 24 hours.


cells compared with 75 at 24 hours; Student’s t-test,p < 0.05) (Fig. 4B,D shows 6 hour time point). These num-bers are not absolute as the greater the tissue swellingresulting from damage, the lower the dead cell count ap-pears within any given unit area. Nevertheless, the signifi-cant reduction in dead cell numbers indicates theneuroprotective activity of Cx43 ASODN treatment.

3.4. Gap junction (Cx43) labeling

Confocal images were viewed of sections taken throughthe optic nerve from the cut edge to the middle of the nerve.Immunohistochemical labeling demonstrated that Cx43signal is sparsely distributed in freshly fixed optic nerve seg-ments. After a 2 hour incubation in OGD solution, Cx43up-regulation was evident at the cut edge of the tissue. ThisCx43 up-regulation gradually increased and peaked ataround 2 days to 3 days post-ischaemia when significantup-regulation is observed. Expression of this protein thengradually declined after day 3. By day 6, the amount of la-bel had returned to a level comparable with that at day 1.No significant difference in Cx43 labeling at the cut edge ofthe nerve (where both control and treated segments haveexperienced damage) was observed between control andtreated nerves at any time.

In ASODN treated nerves, however, the Cx43 up-regu-lation was confined to the cut edges of the optic nerve,whereas the expression of Cx43 was elevated within 3 daysthroughout the entire length of the optic nerve in the con-trol group (Fig. 5A). This temporally specific Cx43 up-reg-

Fig. 5. Montages of three confocal optical slices along the length of day 3optic nerves labeled for connexin43 (Cx43) protein. (A) Cx43 has been up-regulated throughout the control tissue but in (B) its up-regulation islimited to the cut edge (towards the right-hand side of the image) in theCx43 specific antisense oligodeoxynucleotides (ASODN) treated nerves.

ulation in the middle of the optic nerve post-ischaemia wasknocked down by ASODN treatment (Fig. 5B) at all thetime points investigated (1, 2, 3, and 6 days post-ischae-mia). The gel-only control showed no difference to thatof the no-treatment control.

3.5. Co-localization - Cx43 in blood vessel walls

Blood vessels in control tissues showed evidence of dis-integration when labeled for vWF. Mouse anti-Cx43 anti-bodies and vWF antibodies were therefore used tocorrelate Cx43 expression with loss of vascular integrity.After double-labeling of Cx43 and blood vessels, the co-localized area was calculated as a percentage of the bloodvessel area in order to eliminate any initial size differencesin the blood vessels investigated (larger vessels may havea larger area of co-localization).

Analysis of images collected after 1 day in culture revealno significant difference in co-localization between controland Cx43 specific ASODN treated groups. At this time,however, the vascular Cx43 in control nerves became up-regulated and was most prominent after 3 days in culture.Control groups show an approximately 2-fold to 4-fold sta-tistically significant increase in blood vessel-associatedCx43 levels compared to the ASODN treated nerves (Stu-dent’s t-test, p = 0.01). Blood vessel fragmentation wasquantified by calibrating the image and using a line toolto measure the length of blood vessel segments seen usingImageJ software. Assuming that both the control groupand the ASODN treated group had the same number ofblood vessels at time zero, an increase in the number ofblood vessel segments implies vessel fragmentation. Bloodvessel segment numbers in the control optic nerves weregreater than in ASODN treated nerves at days 1, 2, and3 (Table 2); ASODN treated groups, on average, had28% to 50% fewer blood vessel segments in comparisonto the control. There was no obvious difference by day 6.However, the segment lengths in the control optic nervesegments appeared shorter at all times, being relatively sta-ble from days 1 to 3. The blood vessel segment lengths inboth control and ASODN treated nerves, although still dif-ferent, were reduced at day 6, indicating general tissue deg-radation by then in this model.

Table 2The average number of blood vessel segments counted in six sections incontrol and antisense oligodeoxynucleotide treated groups

Number of segments per section

Control ASODN

Day 1 23.5 10.8Day 2 19.2 13.3Day 3 41 29.7Day 6 13.8 18.2

ASODN = antisense oligodeoxynucleotides.

Fig. 6. Glial fibrillary acidic protein (GFAP) labeling of astrocytes in control (A, C) and connexin43 (Cx43) specific antisense oligodeoxynucleotides(ASODN) treated optic nerve segments. Images were collected by taking eight confocal optical slices through 16 lm tissue depth and then producing anextended focus image. After one day in culture, GFAP labeling was (A) slightly higher at the ends of control segments compared with (B) nerves treatedwith Cx43 specific ASODN. By day 2, however, astrocytes in the control optic nerve sections (C) showed a notable level of hypertrophy in both processesand cell bodies, minimally observed in (D) Cx43 ASODN treated nerves indicating a greater inflammatory response in the untreated control nerves. Cellbodies have been circled. Astrocyte activation occurred throughout the control nerves but was restricted to the cut edges in the Cx43 ASODN treatednerves.


3.6. Astrocytic GFAP labeling

After GFAP labeling of astrocytes, data were collectedby taking eight confocal optical slices through a 16 lm tis-sue depth, and making an extended focus projection image.After 1 day in culture, GFAP labeling was slightly higherat the ends of control segments compared with nerves trea-ted with Cx43 specific ASODN (Fig. 6). By day 2, however,astrocytes in the control optic nerve sections showed anotable level of hypertrophy in both processes and cellbodies that was only minimally observed in Cx43 ASODNtreated nerves (circled in Fig. 6C,D), indicating a reducedinflammatory response in the ASODN treated nerves.

After 6 days in culture, a clear decrease in the number ofastrocytes was observed in both control and ASODN trea-ted nerves, suggesting general tissue degradation by thatstage in the model used. Astrocyte activation occurredthroughout the control nerves, but it was restricted to thecut edges in the Cx43 ASODN treated nerves.

3.7. Microglia-isolectin B4 label

No differences were observed between the numbers ofmicroglial cells in the control and ASODN treated groupsat day 1 or day 3. The phenotype of the microglial cellswas, however, very different between the two treatmentgroups. In control groups, by day 3, most cells had differ-entiated into the activated macrophage-phenotype, whilein ASODN treated nerves, more microglia remained inthe small star-shaped inactive form (Fig. 7). This differencebetween activated microglial numbers in control nervescompared to the Cx43 ASODN treated group was signifi-cant at day 3 (average 42 compared with 23 per unit area– Student t-test, p < 0.01).

4. Discussion

This study is the first to evaluate inflammation followingtreatment with Cx43 specific ASODN in an ischaemic optic

Fig. 7. Isolectin B4 labeling of microglial cells in control and the connexin43 (Cx43) specific antisense oligodeoxynucleotides (ASODN) treated optic nervesegments. By day 3, most microglial cells had differentiated into (A) the activated macrophage-phenotype in control groups, whereas in (B) ASODNtreated nerves, more microglia remained in the small star-shaped inactive form. This difference between activated microglial numbers in control nervescompared to the Cx43 ASODN treated group was significant at day 3 (average 42 compared with 23 per unit area - Student t-test, p < 0.01), indicating agreater inflammatory response in the untreated control nerves.


nerve in vitro model. Results demonstrate that applicationof Cx43 ASODN modulated several facets of the inflam-matory process by down-regulating Cx43: (i) reduced tissueswelling; (ii) improved vascular integrity; and (iii) sloweddifferentiation of inflammatory cells. In addition to anoverall dampening of the inflammatory response therewas limited lesion spread, with Cx43 ASODN treatmentrestricting damage to the cut ends of the optic nerve sec-tion. These findings support the concept that ischaemic in-jury to the optic nerve is associated with an up-regulationin the local inflammatory response that is at least partiallyexacerbated by a Cx43 gap junction-mediated bystandereffect.

There is increasing evidence that gap junctions mediatethe spread of cell death following injury by allowing thepassage of death signals via the Cx43 mediated gap junc-tion channels from injured cells to healthy neighbouringcells, precipitating further damage. In the skin, Cx43 hasa major role in modulating the wound healing process.10,18

Cx43 is expressed in the basal epidermis, fibroblasts anddermal appendages. Wound closure is significantly fasterin Cx43 conditional knockout mice19 and in mice whereCx43 specific antisense was applied to the wound to tran-siently down-regulate Cx43 protein expression.10 Cx43knockdown reduced both macroscopic and microscopicevidence of inflammation, which resulted in significantlyless deposition of granulation tissue and subsequently asmaller, less distorted scar.10 Similarly, using Cx43 specificantisense to transiently down-regulate Cx43 protein in theearly stages of wound healing of a partial thickness cutane-ous burn reduced the spread of tissue damage and neutro-phil infiltration around the wound following injury.20

Most of our understanding of the role of inflammationin the CNS comes from studies in spinal cord injury andCNS stroke models. In rodent models of stroke injury,blocking gap junction communication using a non-specificoctanol blocker reduced lesion size,21 In addition, commu-nication through Cx43 gap junctions in in vitro studies canexacerbate cell death in neuronal co-cultures subjected to

oxidative stress.22 In acutely prepared brain slices and incultured astrocytes, the gap junctions remain open duringischaemic or hypoxic conditions.23,24 In spinal cord injurymodels, Cx43 channels are up-regulated and treatmentwith Cx43 specific ASODN (as in the present study) ledto reduced oedema, inflammation and protein leakagefrom vessels, with treated animals showing significantly im-proved behavioural scores on locomotor tests.25

In our in vitro model, the peak of Cx43 up-regulationpersisted for 2 to 3 days post-ischaemia. This appears sim-ilar to previously reported results in peripheral nerve crushinjuries,26 although significant swelling can occur as earlyas within 2 hours in in vivo brain slice experiments underOGD conditions.27 Cell swelling is thought to be causedby cytoplasmic oedema resulting from the opening ofCx43 hemichannels.22,23 Our finding of the onset of swell-ing at 6 hours is comparable with the onset of swelling inthe brain following ischaemia.28 There are no neural cellbodies present in the optic nerve, and swelling must be pre-dominantly within astrocytes or oligodendrocytes. Theexpression of Cx43 in blood vessel endothelial cells mayalso be in hemichannel form, the opening of which wouldlead to endothelial cell death, together with a gap junc-tion-mediated bystander effect, and cause the vessel frag-mentation we observed. Axon injury rapidly activatesmicroglial and astroglial cells that are close to axotomizedneurons, with Cx43 upregulation seen within hours.29 Ourstudy demonstrated similar changes with activation ofmicroglial and astroglial cells in control specimens extend-ing to the middle of the optic nerve sections. However,Cx43 ASODN limited both astrocyte activation and thedifferentiation of microglial cells into the macrophage phe-notype to near the cut edges.

In this study, Cx43 up-regulation was blocked usingunmodified ASODN, which has been shown to reduceconnexin43 protein levels by binding selectively to mRNAsites, thereby preventing protein translation.30 Pluronic F-127 gel provided sustained delivery for the ASODN, whichhas a short half-life owing to breakdown by intrinsic


RNases. Pluronic F-127 gel is a weak surfactant and at30% (w/v) sets as hydroscopic gel at physiologic tempera-ture allowing the continuous release of a reservoir of oligo-nucleotides.11,12,31 ASODN technology was used in thisproject as opposed to other RNA techniques such as smallinterfering RNA (siRNA). Our intention was to transientlydown-regulate Cx43 gene expression for the immediate andshort term, enabling a subsequent return of connexin levelsto normal, rather than a longer term knock-down withsiRNA.32

Despite evidence for the participation of a Cx43 gapjunction-mediated bystander effect under pathological con-ditions, Nakase et al. showed that Cx43 null mice exhibitsignificantly larger infarct volumes.33 The same authorsalso reported that astrocytic Cx43 expression reducesapoptosis and inflammation of neurons during focal brainischaemia.34 While these studies used knockout gene mod-els where connexin levels to not subsequently return to nor-mal, there remains some controversy surrounding the roleof Cx43 in the modulation of the inflammatory responsefollowing injury. The contribution of Cx43 may dependupon cell types, coupling, differentiation, and expressionof other connexin isoforms.35

In conclusion, we investigated the role of Cx43 in anin vitro organotypic culture model of optic nerve damageand ischaemia, with and without the modulation of Cx43expression, by using an antisense oligonucleotide ap-proach. Our findings support the idea that Cx43 ASODNblocks the up-regulation of Cx43, which in turn can reducecell death, optic nerve oedema, activation of astrocytes andmicroglia, and preserve vascular integrity. Together thesemodifications of the inflammatory response resulted in re-duced lesion spread and minimization of the gap junc-tion-mediated bystander effect that often occurs as aconsequence of CNS injury. Future directions presently un-der investigation by our laboratory include the applicationof this new Cx43 regulating technology to an in vivo rodentmodel of ischaemic optic nerve injury (ischaemia and par-tial transection).

Acknowledgements

This work was supported by The Marsden Fund admin-istered by The Royal Society of New Zealand. AuthorCRG acts as a consultant for CoDa Therapeutics, Inc.who hold the patents related to the use of connexin specificantisense for therapeutic purposes and are undertakingclinical trials with connexin specific antisense. CRG is des-ignated as an inventor on patents granted and pendedclaiming the use on connexin specific antisense for the ther-apeutic treatments of humans or animals.

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