Immunohistological intensity measurements as a tool to assess sarcolemma‐associated protein expression

Abstract

(2010) Neuropathology and Applied Neurobiology 36, [265][266][267][268][269][270][271][272][273][274] Immunohistological intensity measurements as a tool to assess sarcolemma-associated protein expression Aims:The quantification of protein levels in muscle biopsies is of particular relevance in the diagnostic process of neuromuscular diseases, but is difficult to assess in cases of partial protein deficiency, particularly when information on protein localization is required. The combination of immunohistochemistry and Western blotting is often used in these cases, but is not always possible if the sample is scarce. We therefore sought to develop a method to quantify relative levels of sarcolemma-associated proteins using digitally captured images of immunolabelled sections of skeletal muscle. Methods: To validate our relative quantification method, we labelled dystrophin and other sarcolemmal proteins in transverse sections of muscle biopsies taken from Duchenne muscular dystrophy and Becker muscular dystrophy patients, a manifesting carrier of Duchenne muscular dystrophy and normal controls. Results: Using this method to quantify relative sarcolemmal protein abundance, we were able to accurately distinguish between the different patients on the basis of the relative amount of dystrophin present. Conclusions: This comparative method adds value to techniques that are already part of the diagnostic process and can be used with minimal variation of the standardized protocols, without using extra amounts of valuable biopsy samples. Comparative quantification of sarcolemmal proteins on immunostained muscle sections will be of use to establish both the abundance and localization of the protein. Moreover, it can be applied to assess the efficacy of experimental therapies where only partial restoration or upregulation of the protein may occur.

Measuring dystrophin—faster is not necessarily better

“…One important aspect is that the original method involves collection of up to 40 data points per section, each corresponding to a muscle fibre, whereas the recent modification involves collecting an average dystrophin intensity of the whole image. For averages or multiple measurements in sections from a manifesting carrier, a clear segregation of measurements is immediately evident using the original method 10 , which is lost with the new method (Figure 1a and b). 2 Similarly, two patients from a recent clinical trial have almost identical levels of dystrophin when assessed using the new Author's pre-print version.…”

“…10 The method uses intensity measurements from fluorescently labelled dystrophin antibodies and spectrin labelling as a normalising factor. The technique greatly advanced dystrophin quantification owing to its sensitivity, requirement for very little sample, capacity to confirm the correct localization of the protein at sarcolemma, and accessibility to most pathology laboratories.…”

Dystrophin as a therapeutic biomarker: Are we ignoring data from the past?

“…Detailed dystrophin quantification using one of these [21] along with clinical correlations in BMD patients clearly indicate that internally deleted dystrophin isoforms have the capacity to confer marked clinical benefits to individuals with DMD [22]. Anthony and colleagues [22] reported that muscle dystrophin expression in BMD patients with a deletion end-point of exon 51 were higher than those in BMD patients whose deletions ended with exon 53.…”

“…With the advent of dystrophin restoring strategies and the need for meticulous evaluation of therapies, improvements in dystrophin detection and quantification have become an imperative, and two groups have published detailed methods for the unbiased quantification of dystrophin immunofluorescent expression [20,21]. Detailed dystrophin quantification using one of these [21] along with clinical correlations in BMD patients clearly indicate that internally deleted dystrophin isoforms have the capacity to confer marked clinical benefits to individuals with DMD [22].…”

“…The sensitivity of current immunofluorescent quantification methods [20,21] should allow reproducible fold-change quantification at even low levels of baseline expression, but whether dystrophin expression is measured by western blotting or by immunohistochemistry techniques, it is logical that fold changes and not fractional increases in dystrophin production above baseline will be needed to ameliorate disease progression in DMD. The minimally sufficient fold change necessary for functional improvement is unlikely to be universally definable; it may differ for different therapies, as it may be dependent upon the induction of the specific dystrophin isoform resulting from therapy, the duration of treatment, and other factors.…”