Teoría de la Comunicación Productiva de Nosnik

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Figure 4.2.2-16. CM-IR in lumbar motoneurones (arrowheads) after axotomy of the nerve to the sartorius (sart) muscle. There appears to be some reduction in immunostaining bilaterally in comparison to the unlesioned motoneurones supplying the iliacus muscle.

The reasons for the paucity of immunostaining in four of the sacral axotomy experiments are not clear. The antibody was found to be somewhat capricious, in spite of maintaining high standards of consistency in the washing and incubation of the tissue, which may account for some of the variability. In addition, the decrease of immunoreactivity resulting from the injury might also have exacerbated the problem. The reasons for the inclusion of other ventral horn cell types in this response are not clear, but could relate to transneuronal effects of axotomy. Thus the decrease of total ventral horn immunoreactivity in those experiments may have been - at least in part - a genuine reflection of the injury-induced changes in CM-IR.

In spite of a low residual sample size, however, the findings of CM-IR decreases were particularly robust and were examined using a variety of statistical procedures, the results of which were all highly significant, and are in agreement with the only other published account of post-axotomy motoneuronal immunoreactivity for CM.

A decrease in CM-IR, but not mRNA, has been reported in axotomised rat hypoglossal motoneurones between the 2"^^ and 21®* day after nerve transection (Dassesse et al, 1998), suggesting a decrease in stored gene product. These authors postulate a post-transcriptional factor such as Ca^+ dependent ubiquitination and degradation to account for the apparent decrease in CM-IR.

The reduction in levels of immunoreactivity in the absence of changes in mRNA levels may alternatively be explained by two hypothetical mechanisms of decreasing antibody access: increased substrate binding or conformational changes. Firstly, in the presence of axotomy-induced increases in [Ca^+]i, the association of CM with its target proteins may be elevated. Secondly, axotomy-

induced elevations or de novo expression of proteins that are involved in the regenerative changes resulting from axotomy may lead to a shift of CM binding tendencies in favour of these novel or up-regulated substrates, altering its conformation. For example, GAP-43, which has been described as a “calmodulin sponge” (Skene, 1990), is bilaterally elevated in the motoneuronal cell bodies and fibres after axotomy (Booth and Brown, 1993; Fiehl et al, 1993). Increased or altered substrate binding by CM could then interfere with the ability of the antibody to detect the antigen due to conformational changes or masking of the antibody binding site.

An additional interpretation of these data may be that CM transcription after axotomy is actively suppressed to reduce the activity of degeneration promoting pathways, although given the breadth of neuronal functions that CM supports, it seems more likely that the CM substrates involved in neurotoxic events, such as CaN and nNOS, would be preferred targets for active and selective suppression. Indeed it may be the case that a primary decrease in levels of CaN and nNOS seen after axotomy (see above) causes a decrease in the utilisation of CM. CM’s other major target protein is CaM kinase, and CaM kinase II has also been found to be decreased after axotomy (Lund and McQuarrie, 1997). Due to decreased utilisation, the surplus CM might then be rapidly eliminated from the cell. In such circumstances, the observed decrease in CM would be a secondary outcome of a decrease of another protein which is of greater relevance to the injury response process.

Such a hypothesis would concur with the findings of the present study. Significant decreases of nNOS-lR and CaN-IR were observed in axotomised pudendal motoneurones. The selective reduction of these two proteins in ON, both of which are normal targets of CM activity, might contribute to the

decrease in the CM-IR of ON as described above. Meanwhile, by the second week after section of n. sartorius, CaN-IR was only slightly decreased and nNOS- IR was elevated. In this case the binding of CM to its target proteins may remain unchanged, and would not be expected to affect levels of stored gene product in the same way. This hypothesis might thus account for the differences between the two segments in injury response.

4.2.3 CaBP- D 28k an d PV

No changes were seen in motoneuronal PV-IR after axotomy. The results for CaBP-D28K-IR are presented below.

4.2.3.I. Pudendal nerve axotomy

The post-axotomy reductions in levels of CaBP-D28K-IR in ON and VL were clear - particularly affecting the fibres running through ON - and highly significant (Fig. 4.2.3-1). An interesting feature of the pudendal axotomy response was the observation of the occasional swollen motoneurone with an eccentric nucleus which had a higher level of immunostaining than surrounding motoneurones (Fig. 4.2.3-2).

One week after pudendal axotomy the reduction is bilateral, the MBL for VL reducing from 5 to 1.88 and that of ON from 4.22 to 2.9. By the second post­ operative week the contralateral MBLs for both VL and ON have returned to normal levels.. A reduction of the MBL for VM is also evident at week one, suggesting a more widespread response for CaBP-D28K than other antigens. In addition, even though the MBL for VL is significantly reduced from normal on both sides of the cord at the first week, the contralateral side (MBL = 2.2) also has a significantly higher MBL than the ipsilateral side (MBL = 1.88, Dunnett’s T3 sig. p. = .044) showing that the effect is still more pronounced ipsilaterally. Table 4.2.3-1 below summarises the confidence intervals for each post-axotomy reduction. Figure 4.2.3-3 gives the MBLs, and Figures 4.2.3-4 - 4.2.3-6, the distributions.

4.2.3.Ü. N. Sartorius Axotomy

After axotomy of n. sartorius, the MBL of both ipsilateral (1.46) and contralateral (1.47) sartorius motoneurones was significantly lower than that of