Pressure ulcer is a complex and significant health problem. Although the factors including pressure, shear, and ischemia have been identified in the etiology of pressure ulcer, the cellular and molecular mechanisms that contribute to the development of pressure ulcer are unclear. This study tested the hypothesis that the early-onset molecular regulation of pressure ulcer involves apoptosis in muscle tissue. Adult Sprague-Dawley rats were subjected to an in vivo protocol to mimic pressure-induced deep tissue injury. Static pressure was applied to the tibialis region of the right limb of the rats for 6 h each day on two consecutive days. The compression force was continuously monitored by a three-axial force transducer equipped in the compression indentor. The contralateral uncompressed limb served as intra-animal control. Tissues underneath the compressed region were collected for histological analysis, terminal dUTP nick-end labeling (TUNEL), cell death ELISA, immunocytochemical staining, and real-time RT-PCR gene expression analysis. The compressed muscle tissue generally demonstrated degenerative characteristics. TUNEL/dystrophin labeling showed a significant increase in the apoptotic muscle-related nuclei, and cell death ELISA demonstrated a threefold elevation of apoptotic DNA fragmentation in the compressed muscle tissue relative to control. Positive immunoreactivities of cleaved caspase-3, Bax, and Bcl-2 were evident in compressed muscle. The mRNA contents of Bax, caspase-3, caspase-8, and caspase-9 were found to be higher in the compressed muscle tissue than control. These results demonstrated that apoptosis is activated in muscle tissue following prolonged moderate compression. The data are consistent with the hypothesis that muscle apoptosis is involved in the underlying mechanism of pressure-induced deep tissue injury.
- bed sore
- muscle damage
pressure ulcer, also named pressure sore, bedsore, or decubitus ulcer, is defined as the localized ulcerated tissues breakdown caused by sustained, unrelieved mechanical pressure in the body-support interface such as with bed, wheelchair, and orthoses/prostheses. The lesion consists of damage of underlying tissues including skin, muscle, and connective tissues in the compressed region. Pressure ulcer is a significant health problem that occurs commonly in individuals with partially or completely diminished mobility such as frail, infirm elderly and patients with neuromuscular or central neurological impairments (e.g., spinal cord injury) (3, 27, 28, 37, 38). The populations particularly vulnerable to pressure ulcer include those of prolonged bedridden, wheelchair-bound, and orthoses/prostheses clients. In addition to the detrimental impacts on the sufferer's activities of daily living and quality of life, pressure ulcer imposes a tremendous burden on social health care costs.
Pressure ulcer is generally classified as superficial or deep ulcers (6). Increasing recent attention has been put on deep ulcer because, compared with superficial pressure ulcer, deep ulcer results in more severe ulcerated damage with a faster rate of development. Deep ulcer arises in the underlying muscle tissues with bony prominence. The wound always develops into late stage severe ulceration when the ulcer becomes visible at the skin layer. This is a considerable problem because this makes the prognosis extremely uncertain and the subsequent clinical treatment very ineffective (2, 9). The characterization of deep pressure ulcer has been revised by the US National Pressure Ulcer Advisory Panel (NPUAP) by introducing the term deep tissue injury, which specifically characterizes this serious form of pressure ulcer typifying by the incidence of “a pressure-related injury to subcutaneous tissues under intact skin” (2, 4, 9, 12).
Currently, there is no effective strategy and intervention in treating and preventing pressure ulcer. The preclusion of the development of effective treatment and prevention is largely due to the lack of understanding on the underlying mechanism of pressure ulcer. Although the main causal factors, namely pressure, shear, and ischemia, have been identified in the etiology of pressure ulcer, the cellular and molecular mechanisms contributing to the development of pressure ulcer are unclear. Muscle tissue has been demonstrated to have low tolerance to mechanical compression and is highly susceptible to respond to external pressure loading (6, 7, 21, 25). This supports the idea that muscle tissue is an important site for initiating pressure ulcer, particularly the deep pressure ulcer or deep tissue injury (12). Because there is a paucity of research in unveiling the cellular signaling and molecular events in muscle tissue during the development of pressure ulcer, the present study aimed to examine whether apoptosis, a highly coordinated, programmed form of cell death, is involved in the early-onset molecular regulation of pressure ulcer in muscle tissue in response to sustained moderate pressure loading. We tested the hypothesis that muscle apoptosis is induced in an experimental animal model that mimicked pressure-induced deep tissue injury.
MATERIALS AND METHODS
Animal and compression protocol.
Female adult Sprague-Dawley rats aged ∼7 mo (294 ± 4 g body wt; n = 8) were used in this study. Rats were housed in pathogen-free conditions at 20°C and were exposed to a reverse light condition of 12:12-h light/dark. They were fed rat chow and water ad libitum throughout the study period. After acclimatization with the housing environment, rats were subjected to an in vivo pressure ulcer-mimicking protocol, as previously described with minor modifications (17). This experimental animal model was adopted because it was shown to induce pressure-related deep tissue injury in muscle tissue in the absence of morphological damage to skin layer, which mimicked the pathology of deep pressure ulcer (17). In brief, animals were firstly anesthetized by one single intraperitoneal injection with mixture of 100 and 10 mg/kg body wt ketamine and xylazine, respectively. Anesthetization was assured by testing the loss of reflex activities and moustache dithering test. Then, a static external pressure of 100 mmHg compression load was applied to an area of 1.5 cm2 in the hair-shaved tibialis region of the right limb of the rats. Compression load was applied by using a controlled, motorized, mechanical indentor in which the magnitude of compression force was continuously monitored by a three-axial force transducer equipped in the load indentor. A laser Doppler flowmetry (DRT4; Moor Instruments, Axminster, UK) with a contact probe (DP1T/7-V2) was used to monitor the blood flow of the compression site as previously described (17). The compression on right limb of the animal lasted for 6 h in each day and was performed on two consecutive days. The left contralateral limb served as intra-animal uncompressed control. Rats were euthanized 20 h after the last session of compression by overdose of ketamine and xylazine. The euthanizing dose was about three- to fourfold of initial dose of ketamine and xylazine. Tissues directly underneath the compression region from both limbs were collected and quickly frozen in liquid nitrogen-cooled isopentane. Frozen tissues were then stored at −80°C for later analyses. All experimental procedures were carried out with approval from the Institutional Animal Ethics Subcommittee of The Hong Kong Polytechnic University. The animal care standards were followed by adhering to the recommendations for the care of laboratory animals as advocated by the American Association for Accreditation of Laboratory Animal Care.
Hematoxylin and eosin staining was carried out to examine the tissue histology. Sectioning of the collected tissues was made perpendicular to the skin to obtain frozen cross section at a thickness of 10 μm in a freezing cryostat at −20°C. The sections were air dried at room temperature and then fixed with 10% formalin (HT-5011; Sigma Aldrich, St. Louis, MO). The sections then underwent a standard staining procedure with Mayer's hematoxylin (MHS-1, Sigma Aldrich) and 1% eosin in CaCl2 (318906, Sigma Aldrich). The examination of the sections was performed by determining the presence of any degenerative histological characteristics such as loss of tightly packing myofiber arrangement, disappearance of angular myofiber contour, increased accumulation of nuclei, and internalization of nuclei in myofibers.
TUNEL and dystrophin staining analysis.
The muscle-related nuclei with apoptotic DNA strand breaks was assessed by double-fluorescent labeling of terminal dUTP nick-end labeling (TUNEL) and dystrophin. A fluorometric TUNEL detection kit was used according to the manufacturer's instructions (11684795910; Roche Applied Science, Indianapolis, IN). In brief, 10-μm-thick frozen sections from control tissues and tissues lying beneath the compressed region were cut in a freezing cryostat at −20°C. Tissue sections were air dried at room temperature, fixed with 4% paraformaldehyde in PBS, pH 7.4, at room temperature for 20 min, permeabilized with 0.2% Triton X-100 in 0.1% sodium citrate at 4°C for 2 min, and incubated with the provided fluorescein-conjugated TUNEL reaction mixture in a humidified chamber at 37°C for 1 h in the dark. Omission of the addition TdT enzyme in the TUNEL reaction mixture on the tissue sections was included as negative control. The sections were then processed with standard immunocytochemical staining procedures to label dystrophin, which aimed to visualize the sarcolemmal membrane of the tissue section. Following the TUNEL labeling, tissue sections were incubated with an anti-dystrophin mouse monoclonal antibody (D8168, Sigma) followed by an anti-mouse IgG Cy3 conjugate F(ab′)2 fragment incubation (C2181, Sigma). The sections were then mounted with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium to visualize nuclei (Vectashield mounting medium; Vector Laboratories, Burlingame, CA). TUNEL- and DAPI-stained nuclei and dystrophin staining were examined under a fluorescence microscope (Biological Research Microscope 80i; Nikon, Tokyo, Japan) with equipped digital camera (DXM 1200c, Nikon). SPOT RT software (Diagnostic Instruments, Sterling Heights, MI) was then used to stack and analyze the images. The numbers of TUNEL- and DAPI-positive nuclei were counted, and only the labeled nuclei that were apparently related to (i.e., under or on) dystrophin staining were counted. Data were expressed as TUNEL index, which was calculated by counting the number of TUNEL-positive nuclei divided by the total number of nuclei (i.e., DAPI-positive nuclei) multiplied by 100. The TUNEL index for each muscle was calculated from four random, nonoverlapping fields at an objective magnification of ×20. It is worth noting that attempts had been made to verify the myogenic identity of the TUNEL-positive labeled nuclei by double staining with TUNEL and anti-MyoD antibody (554130; BD Pharmingen, San Diego, CA and M3512; DAKO, Carpinteria, CA) in the samples. However, we did not identify any MyoD immunopositive nuclei in our examined tissue sections.
Subcellular fractionation and apoptotic DNA fragmentation analysis by cell death ELISA.
A previously described tissue fractionation method was adopted to extract the cytosolic protein fraction followed by the measurement of apoptotic cell death ELISA (22, 29–34). The purity of the protein extract was confirmed by Western blotting using anti-histone H2B (07371; Upstate Biotechnology, Lake Placid, NY) and anti-CuZnSOD antibodies (sc-11407; Santa Cruz Biotechnology, Santa Cruz, CA). The protein content of the extract was quantified in duplicate by Bradford protein assay (Pierce Chemical, Rockford, IL). Cell death ELISA kit (Roche Applied Science) was then used to quantitatively determine the apoptotic DNA fragmentation by measuring the cytosolic histone-associated mono- and oligonucleosomes. Briefly, the cytosolic fraction of muscle tissues was used as an antigen source in a sandwich ELISA with a primary anti-histone mouse monoclonal antibody coated to the microtiter plate and a second anti-DNA mouse monoclonal antibody coupled to peroxidase. The amount of peroxidase retained in the immunocomplex was determined by incubating with 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate). The colorimetric change was monitored at a wavelength of 405 nm by using a microplate reader. Measurements were performed with control and compressed samples analyzed on the same microplate in the same setting. The OD405 reading was then normalized to milligrams of protein used in the assay and presented as an apoptotic DNA fragmentation index.
The protein abundance of apoptotic factors caspase-3 (cleaved fragment), Bax, and Bcl-2 were examined by immunocytochemical staining using fluorescent immunostaining or Vectastain avidin-biotin complex (ABC) detection method (Vector Laboratories) in the control and compressed tissues. The abundance of cleaved/active caspase-3 fragment was used to indicate the activation of effector apoptotic enzyme caspase-3. Before the immunostaining procedure of caspase-3 and Bax, TUNEL was first performed by following the aforementioned procedure to double label both TUNEL nuclei and the caspase-3 or Bax on the same tissue section. The staining of cleaved caspase-3 was carried out using Vectastain Elite ABC peroxidase kit (PK6100, Vector Laboratories) with ImmPACT diaminobenzidine (DAB) peroxidase substrate (SK4105, Vector Laboratories), whereas the fluorescent staining of Bax was mediated by the anti-rabbit Cy3 secondary antibody. Single staining of Bcl-2 was performed using Vectastain ABC alkaline phosphatase kit (PK5000, Vector Laboratories) with Vector Black alkaline phosphatase substrate (SK5200, Vector Laboratories). Briefly, 10-μm-thick frozen sections from the collected tissues were cut in a freezing cryostat at −20°C and were then air dried at room temperature and fixed in 10% neutral buffered formalin. For staining of caspase-3 and Bax, sections first underwent TUNEL procedure. Sections were then incubated in 3% hydrogen peroxide in PBS to quench the endogenous peroxidase activities followed by an avidin/biotin blocking procedure (SP2001, Vector Laboratories) (for caspase-3 only). Sections were then blocked in 5% normal serum (goat serum for caspase-3 and Bax staining; horse serum for Bcl-2 staining) in PBS. After washes in PBS, sections were incubated with an anti-cleaved caspase-3 rabbit polyclonal antibody (9661; Cell Signaling Technology, Beverly, MA), anti-Bax rabbit polyclonal antibody (ab7977–1; Abcam, Cambridge, MA), or anti-Bcl-2 mouse monoclonal antibody (sc7382, Santa Cruz) followed by a biotinylated anti-rabbit antibody (for cleaved caspase-3), a biotinylated anti-mouse antibody (for Bcl-2), or an anti-rabbit Cy3-conjugated antibody (C2306, Sigma) (for Bax) incubation. Negative control experiments were done by omitting the primary or secondary antibody from the tissue sections. Sections for Bax staining were then mounted with VectaShield mounting medium containing DAPI solution (H-1200, Vector Laboratories). For caspase-3 and Bcl-2 staining, sections were then incubated with prepared ABC-peroxidase reagent and colorimetrically detected by incubating with DAB peroxidase substrate (for cleaved caspase-3) or with ABC-alkaline phosphatase reagent followed by Vector black alkaline phosphatase substrate incubation (for Bcl-2 staining).
Real-time RT-PCR gene expression analysis.
Total RNA was extracted from the frozen control and compressed tissues in ice-cold TriReagent (Molecular Research, Cincinnati, OH) on the basis of the guanidine thiocyanate method. Extracted RNA was quantified at 260 nm, and the 260/280 ratio was examined to confirm the purity of RNA. Reverse transcription was then performed with Superscript III reverse transcriptase kit (Invitrogen Life Technology, Carlsbad, Ca) to generate cDNA according to the manufacturer's recommendations. Real-time PCR was performed in TaqMan Master Mix, primer and TaqMan probes, cDNA and RNase, and DNase free water on an ABI7500 real-time PCR machine (Applied Biosystems, Foster City, CA). Primers and probes (TaqMan Assays) were designed at Applied Biosystems while the PCR amplification were verified and the threshold for kinetic detection was set to occur over linear amplifications. GAPDH was included as internal housekeeping control gene. GAPDH was used as internal control because it has been demonstrated to be stably expressed in skeletal muscle in response to physical stress (16), and our data indicated that the mRNA content of GAPDH did not alter with the present pressure-loading protocol. All samples were run in duplicate, with control and compressed samples run on the same plate. The relative quantification of gene expression was calculated using the comparative Ct method (19). Briefly, Ct value indicates the cycle number at which a significant increase in ΔRn (i.e., fluorescence) is first detected. A sample will be considered positive at the cycle in which the change in the fluorescence exceeds an arbitrary threshold value. Comparative Ct calculations for genes of interest were expressed relative to the GAPDH signal generated from the same cDNA sample. The average Ct for the gene of interest was subtracted from the corresponding averaged GAPDH Ct value for that sample to give a ΔCt value. ΔΔCt values were achieved by subtracting the control ΔCt value from the compressed ΔCt value. Data were expressed as the fold differences over controls normalized to the housekeeping gene.
Data were presented as means ± SE. Paired t-test was used to examine differences between control and compressed groups. Level of statistical significance was accepted at P < 0.05.
According to our histological analysis, muscle tissue following the compression procedure generally demonstrated the characteristics of degeneration. The muscle tissue in the region close to the bone particularly showed apparent degenerative characteristics. These comprised rounding-shaped myofibers, accumulated number of nuclei in the interstitial space, and centralized nuclei in the myofibers of the examined muscle tissue cross sections (Fig. 1). Consistent with the previous report, the morphological damages were not observed at the cutaneous tissue in the compressed tissue despite the presence of muscle tissue-damaging characteristics (17). Furthermore, the blood flow as measured by Doppler flowmetry in the compression area was found to be reduced by ∼50%, and this was in agreement with the previous report (17). These data illustrated that the adopted experimental animal model operationally did not result in complete occlusion of local blood flow in the compressed region.
Apoptotic nuclear DNA breaks in muscle tissue were measured using the technique of TUNEL staining (Fig. 2, A–H) and were expressed as TUNEL index (Fig. 2I). Immunofluorescent staining of sarcolemmal dystrophin was performed together with TUNEL staining aimed to facilitate the identification of muscle-associated nuclei. Our TUNEL/dystrophin staining analysis indicated that there were 4.1 ± 1.0% TUNEL-positive apoptotic muscle-related nuclei in the compressed muscle tissue. The percent of TUNEL-positive nuclei was significantly greater in compressed muscle compared with control tissue (P < 0.05) (Fig. 2I).
Apoptotic DNA fragmentation index.
The result of our quantitative DNA fragmentation analysis by cell death ELISA was consistent with the TUNEL analysis, indicating that there was an ∼3.2-fold elevation in the extent of apoptotic DNA fragmentation in the compressed muscle relative to the control muscle (Fig. 3).
Immunoreactivity of cleaved caspase-3, Bax, and Bcl-2.
The activation of caspase-3, an essential effector caspase, in the muscle tissue was evaluated by immunocytochemically detecting the protein abundance of the cleaved caspase-3, which is the active form of caspase-3. Our double staining demonstrated remarkable increase in the immunoreactivity of cleaved caspase-3 (Fig. 4, E and F) in the presence of TUNEL-positive nuclei (Fig. 4, A–D) in the compressed muscle tissue. This suggested that caspase-3 was activated in response to the present experimental protocol with sustained moderate compression. The protein abundance of apoptotic factors Bax and Bcl-2 was examined by immunocytochemical analysis. The result of the TUNEL/Bax double labeling showed that the immunoreactivity of Bax was increased (Fig. 5, E and F) in the presence of TUNEL-positive nuclei (Fig. 5, A–D) in the compressed muscle tissue. It is observed that both the myofibers with and without TUNEL-positive nuclei showed increased immunoreactivity of cleaved caspase-3 and Bax in our examined compressed muscle tissue (Figs. 4 and 5). Our immunocytochemical analysis indicated that the protein abundance of Bcl-2 was elevated in the compressed muscle tissue compared with the control tissue (Fig. 6). The detected immunoreactivity of Bcl-2 was generally distributed in all the myofibers in our examined compressed muscle tissue (Fig. 6).
Apoptotic factor gene expression.
The mRNA contents of caspase-3, caspase-8, caspase-9, Bax, and Bcl-2 were estimated by real time RT-PCR analysis. It was found that the mRNA contents of caspase-3, caspase-8, and caspase-9 of the compressed muscle tissue were elevated by 7.7 ± 2.2-, 3.1 ± 1.0-, and 2.1 ± 0.5-fold, respectively, relative to the control uncompressed muscle tissue (P < 0.05) (Fig. 7). The mRNA abundance of Bax of the compressed muscle tissue was increased by 2.6 ± 0.6-fold (P < 0.05) compared with the control tissue, whereas no significant difference was found in the mRNA content of Bcl-2 in the compressed and control muscle tissues (Fig. 7).
Deep pressure ulcer comprises a serious health problem, which is currently lacking effective regimens in treatment and prevention. Understanding the signaling events that regulate the initiation and development of pressure ulcer should provide valuable information in identifying potential therapeutic targets for effective treatment and prevention. In the present study, we provided novel data that revealed the underlying apoptotic regulatory signaling mechanisms of pressure-induced deep tissue injury in a reproducible experimental animal model. The currently used experimental model was adopted from Kwan and coworkers (17), in which a static moderate pressure compression of 100 mmHg or 13.3 kPa was applied over the tibialis region of unilateral limb of the rats for 6 h per day on two consecutive days. Consistent with the findings reported by Kwan and colleagues (17), the presence of degenerative characteristics was demonstrated in the underlying muscle tissue but not cutaneous tissue in the examined compressed tissues. As indicated by the Doppler flowmetry measurement, the applied compressive pressure was shown to result in about 50% reduction of blood flow in the underneath blood vessels in the compressed region (17). With this experimental setting, which resembled the pressure ulcer situation and initiated the pressure-induced deep tissue injury, we showed the activation of apoptotic program by demonstrating increase in TUNEL-positive apoptotic muscle-related nuclei, increase in apoptotic DNA fragmentation, elevations of cleaved caspase-3, Bax, and Bcl-2 protein contents, and increases in mRNA contents of caspase-3, caspase-8, caspase-9, and Bax in the affected muscle tissue in response to sustained moderate compression.
Pressure ulcer can be initiated in cutaneous or deep tissues. For the cutaneously initiated pressure ulcer or superficial type of pressure ulcer, the tissue destruction process begins in the dermal layer and progresses toward the deeper tissue. The resulting ulceration is always visible and easily detected even in the early stage and thus makes the subsequent therapy efficient. In contrast, deep pressure ulcer or deep tissue injury is problematic because the process of the ulceration initiates in deep layers of tissues and progresses unnoticeably under the intact cutaneous layer (6, 12, 35). This eventually causes severe and extensive tissue damage, which results in serious infectious morbidities and mortalities (1, 2, 12). Research employing the techniques of computed tomography, ultrasound, and other biochemical approaches have been conducted with the aim to achieve better diagnosis of the deep pressure ulcer (10, 13, 23, 24). However, the underlying mechanisms responsible for the initiation of deep tissue injury are not known, and this mechanistic information would be necessary when searching for effective means for the deep ulcer treatment and protection. Nevertheless, whereas most of previous pressure ulcer research focused on the skin tissue, increasing attentions have been put on the etiological role of muscle tissue in deep pressure ulcer because of the observations that muscle tissue has a lower tolerance to mechanical compression and higher susceptibility to external pressure compared with other soft tissues such as skin (6, 7, 12, 21, 25). Although there is still a paucity of study addressing the cellular mechanism and molecular events in muscle tissue in deep pressure ulcer, few influential studies have been conducted to demonstrate the potential important role of skeletal muscle in pressure ulcer (7, 17, 21, 25). In agreement with the findings of these studies, the present study also clearly demonstrated that the pathohistological damage in muscle tissue can be induced by sustained moderate compression in the absence of any visible skin damage. Our results confirmed that muscle tissue plays an important role in the pathogenesis of pressure ulcer, and it warrants additional research in fully understanding the response of muscle tissue to pressure, particularly at the very early stage in which none of any tissue damage has been initiated.
Apoptosis is a highly organized form of cell death that involves tight regulation via apoptotic signaling pathways. Although the apoptotic regulatory pathways have not been thoroughly investigated in muscle tissue in pressure ulcer, apoptotic DNA fragmentation as detected by DNA nick translation assay or YO-PRO-1 fluorescent dye staining in response to compression or hypoxia has been reported, at least in the in vitro setting of skeletal muscle cell culture (11, 36). In the present study, we further demonstrated the incidence of apoptotic DNA fragmentation and the corresponding apoptotic signaling following sustained moderate compression. A merit of the present findings is that our results were obtained in an in vivo-controlled pressure-induced deep tissue injury experimental setting, which comprised the intact internal tissue environment. Contrary to in vitro setting, our in vivo experimental setup comprised the considerations of the intrinsic factors and the interactions among tissues in an individual in response to pressure compression. Together with the previous in vitro findings (11, 36), our results substantiated the potential role of apoptosis in deep tissue injury and have elaborated the corresponding apoptotic signaling pathways. In regard to the cellular death in pressure ulcer, it has been suggested that apoptosis may come before necrosis in the development of pressure ulceration although the topic of muscle apoptosis has not been systematically explored in pressure ulcer (26, 35). Apoptosis is regulated by specific apoptotic factors that coordinate the sequential apoptotic events. Indeed, reversibility of the destructive process is possible depending on the stage of the cell death development. As our data demonstrated that the Bcl-2 family protein signaling and activation of caspase-3 pathway are involved in the underlying mechanism of pressure-induced deep tissue injury, this suggested an opportunity that the therapeutic alternative can be designed by targeting these signaling pathways. Because apoptosis is a highly controllable programmed form of biological process, one can hope to modulate or even cease the developmental process of pressure ulcer by steering the corresponding apoptotic signaling. Additional studies are warranted to address this striking research topic.
In addition to the elevations of TUNEL-positive nuclei and apoptotic DNA fragmentation, activation of caspase-3, and increases in Bax and Bcl-2, we found that there was an increase in the transcript content of caspase-8 in the compressed muscle tissue relative to the control. Caspase-8 is an initiator caspase associated with the upstream cellular signaling of the cell death receptor apoptotic pathway (5). This finding suggested the involvement of this specific apoptotic pathway in the pressure-induced deep tissue injury. It is worth noting that the incidence of inflammation-related degenerative characteristics like the accumulated nuclei in the interstitial space of the muscle tissue of the compressed sample was consistent with the caspase-8 data, suggesting the involvement of death receptor pathway because inflammatory factors including TNF-α and Fas have been shown to induce death receptor-mediated apoptosis (15, 18). Nonetheless, the precise relationship between the activated apoptosis and inflammation in pressure-induced deep tissue injury remains to be further elucidated. Additional research is warranted to completely dissect the potential role of death receptor-mediated apoptotic signaling pathway in the etiology of pressure ulcer. Collectively, our data in apoptotic factors suggested that both the death receptor-mediated and mitochondria-mediated apoptotic pathways may be involved on the basis of the caspase-8 and caspase-9/Bcl-2 family protein results, respectively. According to the idea of caspases cascade, different initiator caspases are recruited specifically in different apoptotic pathways, and the signals finally converge on the common effector caspases (8). Our data were consistent with this cascade concept by demonstrating that the mRNA contents of initiator caspase-8 and caspase-9 were increased by two- to threefold, but the effector caspase-3 was increased by over sevenfold in the compressed muscle tissue relative to the control. Although our findings were limited by the lack of the other caspases (e.g., caspase-6/7 and caspase-12), these data generally supported the idea that the apoptotic signal at the level of effector caspase is amplified by merging the signals from initiator caspases.
Although our immunocytochemical staining demonstrated the overall increase in caspase-3 and Bax in the examined myofibers of muscle tissue following compression, the parallel TUNEL analysis indicated that the immunoreactivity of caspase-3 and Bax did not necessarily correspond to the detection of TUNEL-positive nuclei. The absence of TUNEL nuclei in the myofibers demonstrating caspase-3 or Bax immunoreactivity was indeed not impossible. The apoptotic factors caspase-3 and Bax play a role in coordinating the apoptotic breakdown of DNA strands, which is an outcome of apoptosis. It would be reasonably expected that some of the myofibers currently showing immunoreactivity of caspase-3 or Bax might ultimately be detected with TUNEL nuclei if the compression duration is prolonged. Nonetheless, the present study might have missed the time point at which caspase-3/Bax immunoreactivity and TUNEL nuclei simultaneously exist. Our immunocytochemical analyses indicated that there was a marked increase in proapoptotic Bax protein content, but the antiapoptotic Bcl-2 protein abundance was also found to be elevated. We interpreted the observation that antiapoptotic Bcl-2 that was upregulated in the compressed muscle tissue might be caused by adaptations of antiapoptotic events in an attempt to reduce the proapoptotic environment in response to the pressure-induced deep tissue injury, particularly in the early stage. Indeed, there have been previous reports that demonstrated the concomitant activation of anti- and proapoptotic signaling events. For instance, the increase in antiapoptotic Bcl-2 has been shown with the activation of apoptosis during muscle denervation, muscle disuse, and sarcopenia (20, 22, 30, 32, 33). The presently observed response of antiapoptotic Bcl-2 might have contributed to a compensatory action to adapt and counteract the increased apoptotic effect during compression. For the RT-PCR analysis in the present study, it is noted that some nonmuscle cell types such as smooth muscle cells, endothelial cells, nerve cells, and fibroblasts were included in the muscle tissue homogenates that we used to extract RNA to study the gene expression of apoptotic factors. Nevertheless, we interpreted that the observed significant changes of the mRNA content of the apoptotic factors were primarily attributed to the muscle cells. It is not likely that the striking changes of the mRNA contents in this study were the result of the major contributions by nonmuscle cells because of the relatively small proportions of these cell populations compared with muscle cells. Nonetheless, our data did not permit us to rule out the possibility that the nonmuscle cell populations might have responded to the present compression-loading protocol.
In regard to the TUNEL staining, there might be possible limitations in illustrating the exact identity of apoptotic myonuclei. It is noted that the myogenic identity of labeled nuclei was primarily estimated on the basis of dystrophin staining. It would be ideal if the exact myogenic identity of the apoptotic labeled nuclei could be authenticated by staining with muscle-specific antigen such as MyoD. However, our attempt at MyoD staining did not reveal any immunopositive nuclei in the compressed muscle tissue samples examined. Nevertheless, the nuclear apoptotic event as demonstrated in this study was supported by both TUNEL staining and apoptotic cell death ELISA. A static moderate pressure compression of 100 mmHg was applied for 6 h per day on two consecutive days to induce deep tissue injury in the present study. It is worth noting that the resulting muscle pathology was initiated in a condition with 50% reduction of blood flow but was not completely ischemic. It would be interesting to compare the present results to the findings obtained from complete ischemia. Although the experimental protocol of our study was not exactly the same from the study conducted by Hatoko and colleagues (14), our findings generally shared the similarities in the results that apoptotic signaling responded to 6 h of muscle ischemia. By clamping the femoral blood vessels, ischemic condition was produced in rat gastrocnemius muscle (14). They reported that the protein level of p53, p21WAF-1, and Bax, as determined by Western immunoblotting, was not changed immediately after 6 h of ischemia alone but was gradually increased during the subsequent 12–72 h of reperfusion (14).
In conclusion, our results demonstrated that apoptosis is activated in muscle tissue in the early stage of deep tissue injury during prolonged moderate compression. This implied that muscle apoptosis may have a role in the underlying mechanism of the development of pressure-induced deep tissue injury. As agreed by the delegates of the NPUAP, the underlying mechanisms of deep tissue injury require more investigations (12, 35). The precise cellular signaling responsible for the pathogenesis of pressure ulcer should be fully revealed by future research, which may lead to the identification of the molecular targets for developing novel treatment and preventive tools. The present findings confirmed that muscle tissue is of high clinical concern as the site for initiating tissue destruction in response to sustained moderate compression. Our findings also provide insight that muscle apoptotic signaling events could be the potential targeting pathways for developing therapeutic and preventive alternatives for pressure ulcer.
This study was supported by The Hong Kong Polytechnic University Research Funds A-PH69 and A-PA7N.
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