Sobre la oralidad y sus elementos

plaque count or total Connexin43 gap junction plaque area in the bystander region (Table 5). Plaque counts in the injured region of the antisense oligodeoxynucleotides treated tendons were significantly higher compared with group M (P=0.0066), group P (P=0.0526) and Group S (P=0.0339) (Table 5, Figure 15). The Connexin43 gap junction plaque total area was not significantly different. The finding may be due to fragmented gap junction plaques resulting in a larger number of small plaques but no difference in total Connexin43 gap junction plaque areas, compared with the other treatment groups. This lack of reduction in Connexin43 gap junction plaque number was unexpected and contradicts the published data on antisense oligodeoxynucleotides treatment (Becker et al., 1999, Qiu et al., 2003, Mori et al., 2006, Cronin et al., 2006). The previous studies using Connexin43 antisense oligodeoxynucleotides were performed in rodents and on

porcine and human cells. Whilst the sequence of the murine antisense oligodeoxynucleotides is the same for sheep and horses it is possible that the tertiary folding structure is different between species and coded for at a different site, and that this influencedthe effectiveness of the antisense oligodeoxynucleotides.

Gap junction protein knockdown begins with a reduction in size of gap junction plaques followed by a reduction in number of plaques as cytosolic stores are exhausted (Cronin et al., 2006). This and the Connexin43 half-life of 1.5-2 hours may explain why the first time points at which gap junction counts are significantly reduced in published data are within 4-24 hours (Cronin et al., 2006, Law et al., 2002, Mori et al., 2006, Brandner et al., 2004) and are dependent on tissue type (Becker et al., 1999). The time points of this study (2 – 4 hours) may therefore represent an early effect of antisense oligodeoxynucleotide treatment on connexin gap junction plaque morphology within the lesion site itself that has not yet been reported. Reverse transcriptase polymerase chain reaction to confirm down-regulation of expression and following some animals for a longer time period would enable further investigation of this.

Following wounding whilst gap junction cell-cell coupling is reduced, surface hemi- channel levels are increased in both cardiac muscle and astrocytes (Smyth et al., 2010 ). These are new data reported following the completion of data collection and processing in the current study. Differentiation between gap junctions and hemi channels was not attempted. Intuitively the hemi channels are likely to be smaller than the gap junctions and this could be another explanation for the increased number of smaller areas of staining as hemi channels move to the membrane before expression is down-regulated. In this model the antisense oligodeoxynucleotides were delivered in pluronic gel to act as a slow release reservoir to overcome the effect of serum breakdown. The rate of

dissolution of the pluronic gel is related to the concentration of the pluronic in the gel. The rate and duration of exposure of the tissue to the antisense oligodeoxynucleotides and the antisense oligodeoxynucleotides to serum breakdown is in turn related to the dissolution of the pluronic gel. The knockdown of Connexin43 expression is achieved by sustained exposure of the tissue to the antisense oligodeoxynucleotides. The 24% gel has been a successful delivery vehicle for antisense oligodeoxynucleotides in other tissues (Cronin et al., 2006) It is possible that this percentage was not ideal in tendon tissue due to high serum concentration in response to injury. A higher concentration gel may be required for effective delivery to tendon tissue.

A further reason that the gap junction plaque count remained high in the tendons treated with antisense oligodeoxynucleotides may have been that the Connexin43 antisense oligodeoxynucleotides may have promoted increased survival of tendon and inflammatory cells in the injured area due to a reduction in bystander effect. Tendon fibroblasts and inflammatory cells were the predominant cell types present in the sections and both express Connexin43 with inflammatory cells having prominent Connexin43 levels (Zahler et al., 2003). The overlap of the cells made individual cell identification and counts impractical and no cell-specific labelling was performed to differentiate between the cell types.

In summary, the factors that could have potentially contributed to the apparent failure of antisense oligodeoxynucleotides to decrease Connexin43 gap junction plaque levels include; a) the potential inflammatory effect of injecting a volume of material (pluronic gel) into the tendon b) the antisense oligodeoxynucleotide dose and product used, c) increased survival of cells, and d) the presence of serum in the lesion. Further studies would be required to address these questions.

Figure 15 - Haematoxylin and Eosin and confocal images

a) Haematoxylin and Eosin stain of tendon from GA control sheep 5. (Bar = 200 μm)

b) Confocal image of tendon from GA control sheep 5 nuclei stained with DAPI (blue) and connexins with Alexa 568 (red). (Bar = 25 μm)

c) Haematoxylin and Eosin stain of tendon from sheep 273 ( injury model)

d) Confocal image of tendon from sheep 273 (injury model) nuclei stained with DAPI (blue) and connexins with Alexa 568 (red)

e) Haematoxylin and Eosin stain of antisense treated tendon from sheep 298

f) Confocal image of antisense treated tendon from sheep 298 nuclei stained with DAPI (blue) and connexins with Alexa 568 (red)

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External factors prevented follow up of the sheep for a longer time, which would have been preferable as most of the data on down-regulation of connexins by antisense oligodeoxynucleotides shows significant down-regulation at 6-8 hours. Also, the sheep were not recovered from the anaesthetic so the injured tendons were not subjected to weight-bearing and loading. This was not ideal as loading is thought to affect lesion expansion (van Schie et al., 2009). A progression of this study would ideally allow postural loading of the tendons after recovery from anaesthesia and examination of the tendons at later time points, for example at 24 h, 5 and 7 days post injury.

External factors also prevented ultrasound imaging of the lesions, the common clinical method of monitoring tendon lesions. This is less of an issue in this acute injury period as significant changes in mild lesions can take a week or more to be visible (De Grandis et al., 2012).

The large sheep to sheep variation in gap junction counts inthis short time-course study resulted in a wide range of values within each group and may have overshadowed smaller variations between groups (Table 5). A larger sample size, intra-animal controls and a longer time course may alleviate this problem. The use of a pressure gauge to measure the force of the thrusts of the stylet would standardise the creation of the lesions and help reduce the sheep to sheep variation further.

Quantification of the effect of anti-sense oligodeoxynucleotides on the spread of the lesion could indicate the likely efficacy as a therapeutic agent. Prognosis of recovery following tendon injury is very dependent on the original lesion size and therefore anything that can block lesion spread will potentially improve healing. The inhalational anaesthetic may have had an influence on lesion spread due to its gap junction blockade effects so monitoring for longer and post recovery would again have been ideal.

Sectioning the tendons through the cyanoacrylate glue and submitting samples for electron microscopy to determine if a good seal had been achieved would have confirmed that only intrinsic repair would occur in this model. Injecting into the tendon lesion with a dye to see if egress occurred at the stab incision would also confirm a seal had been created.