Fig. 1
Immunohistochemical analysis with nuclear factor kappa B p65 antibody. Anterior margin of a massive rotator cuff tear. Activated tenocytes. 20×
Neoangiogenesis evaluation was scored as absent or present (multifocal or diffuse).
NF-kB p65 immunostaining was either cytoplasmic (not activated protein) or nuclear with or without cytoplasmic staining (activated protein). The specific immunoreactivity was investigated in endothelial cells, tendon fibroblasts, and synoviocytes. A semiquantitative assessment of immunostaining was expressed as: score 0 (not activated protein); score 1 (nuclear with or without cytoplasmic staining). In case of adulterated sample, the result was not considered; therefore, some cases were missing.
A quantitative assessment of immunostaining was also executed and was expressed as: not assessable, absent, unifocal, multifocal, widespread. However, we decided not to analyze these results because sample size is not large enough to permit an analysis at such a level of detail and because the expression of the p65 may vary in the same section, making the quantification of p65 difficult. Because no other studies were performed with the aim of establishing if NF-kB is present on rotator cuff tear margins, we considered more appropriate to focus our study on NF-kB localization rather than on a quantitative analysis, such as PCR.
Data were submitted to statistical analysis.
The results of our study are summarized in Figs. 2 and 3. These figures show the frequency of positive and negative responses to p65 and to angiogenesis, respectively, for each type of cuff tear (small, large, and massive) and in the different tissues analyzed (margins of rupture, bursa, or healthy tendon).
Fig. 2
Graphs representation of frequency of positive and negative responses to p65
Fig. 3
Graphs representation of frequency of positive and negative responses to angiogenesis
Figure 4 refers to the percentages of the positive responses to p65 factor. It shows that the presence of the activated p65 increases with the increasing of the tear size. This tendency was observed in the anterior and posterior margin of the cuff tear and in the subacromial bursa. Furthermore, the activated p65 factor was not present in the subscapularis tendon of patients with small cuff tear, but its presence progressively increased in the tendons of patients with large and massive cuff tears.
Fig. 4
Percentages of the positive responses to p65 factor. Differences obtained collapsing percentages of the anterior margin to those of posterior margin, respect to controls, resulted statistically significant (chi square = 7.66, p = 0.02)
Analogously, we observed that neoangiogenesis grows with the increasing of the cuff tear size and that this tendency occurs in the anterior and posterior margins of the cuff tear and in the bursa (Figs. 5 and 6a, b). Unlike as it was registered for the presence of the activated p65 factor, in the subscapularis tendon the neoangiogenesis resulted equally present in the different sized cuff tears.
Fig. 5
Percentages of the positive responses to angiogenesis. Differences obtained collapsing percentages of the anterior margin to those of posterior margin, respect to controls, resulted statistically significant (chi square = 8.03, p = 0.02)
Fig. 6
Neoangiogenesis in an anterior margin of a massive rotator cuff tear; HE 20× (a) and immunohistochemical analysis with nuclear factor kappa B p65 antibody; 20× (b)
Although results point out that the percentage of positive responses to p65 activated factor grows as the seriousness of the cuff tear increases, there was no statistically significant difference between small, large, and massive tears, if we consider separately the anterior (chi square = 2.77, p = 0.250) and the posterior margin of the rupture (chi square = 5.24, p = 0.073). However, when we globally analyzed the results obtained for both margins, without distinguishing the anterior and posterior margin, the difference was significant (chi square = 7.66, p = 0.02).
For the bursae and the healthy tissue, the chi squared test may not be valid because cell frequencies were too small even though the differences that emerged among small, large, and massive tears relative to presence of the activated p65 resulted significant (bursae: chi square = 7.03, p = 0.03; healthy tissue: chi square = 9.2, p = 0.01).
The difference in angiogenesis observed in three types of cuff tears resulted statistically significant for the anterior margin (chi square = 6.17, p = 0.046) but not for the posterior margin (chi square = 2.39, p = 0.3). Again, when we globally analyzed the results obtained for both margins, without distinguishing the anterior and posterior margin, the difference was significant (chi square = 8.03, p = 0.02).
For the bursae and the healthy tissue, the chi square test, used to investigate the difference relative to angiogenesis in the three sizes of cuff tear, may not be valid for the same reasons explained above; however, in this case, the test was not significant (bursae: chi square = 4.4, p = 0.11; healthy tissue: chi square = 0.03, p = 0.98).
The Spearman test indicates that p65 and neoangiogenesis are correlated in spite of the dimension of the cuff tear. The correlation was present when we globally considered the results emerged by all the examined tissues (ρ = 0.299, p = 0.0001) and also in the case when we only considered the anterior and posterior margins of the tears (ρ = 0.236, p = 0.009) or the bursae (ρ = 0.429, p = 0.0006).
Two main results emerged from our study: (1) the presence of the activated p65 factor (one of the five transcription factors that compose the NFkB) on the margins of the tendinous rupture increased with the increasing of the rotator cuff tear size; (2) activated p65 and neoangiogenesis were correlated in spite of the dimension of the cuff tear.
Three hypotheses could explain the first result:
- 1.
NFkB activation induced by tissue apoptosis. Strong support for a protective role for NFkB in apoptosis came from some studies [15–18]. Apoptosis, or programmed cell death, is a physiological process that contributes to control cell population [19]. Excessive apoptosis was observed in degenerative changes of joints [20] and in rheumatoid arthritis [21]. This phenomenon has been attributed to tissue ischemia [22, 23], hypoxia [24], free radical generation [25, 26], and nutritional imbalances [27]. However, it may be possible that the mechanism responsible for the beginning of the apoptosis is a combination of factors [28]. Therefore, our hypothesis was that NFkB may lessen, above all in massive rotator cuff tears, progression of tendinous degeneration keeping the other tendinous cells from death. In fact, Rylei et al. [5] have stated that loss of cellular activity and decreased extracellular matrix synthesis are causes of tendon degeneration. Similarly, Yuan et al. [28] believe that the reduced number of functional fibroblast/fibrocytes may contribute to impaired collagen metabolism culminating in rotator cuff degeneration. The authors have also observed that the percentage of apoptotic cells in the superior cuff tear (34 %) was significantly higher than that revealed in the controls (13 %). Furthermore, they added that there was no correlation between the proportion of apoptotic cells and the size of rotator cuff tear. This affirmation seems to be in contrast with our study because we have observed that NFkB on the margins of the tendinous rupture increases with the increasing of the rotator cuff tear size. However, the same authors considered the possibility that their results were not significant because of the relatively small number of patients (only 25). Furthermore, cuff tears size was not mentioned.Related posts:
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