Age-dependent changes in the transverse carpal ligament and median nerve: a cadaveric histological and biomechanical study

Specimen

One hundred TCL specimens—50 from right hands and 50 from left hands—were obtained from 12-month formalin-embalmed cadavers (21 males, 29 females), aged 40–93 years, without lesions or pathologies in the hands or wrists. Information regarding systemic medical conditions such as diabetes, amyloidosis, or neurodegenerative diseases was not available due to limitations of cadaveric donation records. However, all specimens were grossly examined to exclude visible pathology or injury of the hand and wrist. These cadavers were provided by the Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, between May, 2022 and December, 2024. Written informed consent was personally signed by all donors for educational and medical research purposes. All cadaveric data were fully anonymized prior to being accessed for this study. The study protocol was approved by the Siriraj Institutional Review Board (SIRB), Faculty of Medicine Siriraj Hospital, Mahidol University under an SIRB exempt research (SIRB No. 298/2565 (Exemption)).

Mechanical test

After removing the skin, subcutaneous fat, and fascia from the wrist and palm of each cadaveric forearm, the TCL was exposed. A MyotonPRO device (Myoton AS, Estonia) was then employed in situ to measure four non-invasive parameters detailing tissue properties (Prantil et al., 2012). Specifically, stiffness (dynamic stiffness (N/m)) indicates the tissue’s resistance to an external force that changes its shape. Elasticity (logarithmic decrement) represents how quickly oscillations diminish within the tissue, where higher values indicate lower elasticity. Relaxation time (mechanical stress relaxation time (ms)) is the duration for the tissue to return to its original shape once the external force is removed, with lower values indicating more rapid elastic recoil. Finally, creep (ratio of relaxation and deformation time) describes the gradual deformation of the tissue under a constant load over time; lower creep values denote greater resistance to shape change. Measurements were obtained by positioning the MyotonPRO probe perpendicularly at three distinct sites on the TCL—proximal, central, and distal—all located above the median nerve. The proximal and distal sites corresponded to the respective edges of the TCL, while the central site lay midway between them. Each location was measured three times, and the average of these repeated measurements was used for data analysis.

Histological study

For histological analysis, specimens from the youngest (<60 years, n = 7) and oldest (≥80 years, n = 17) age groups were selected to emphasize age-related differences. This approach was chosen to maximize contrast between extreme age groups. Following the biomechanical tests, each TCL and carpal tunnel contents were excised and immersed in 10% formalin for 24 h at room temperature. Subsequently, each ligament was divided into proximal, middle, and distal segments, rinsed in running tap water overnight, and dehydrated in ascending concentrations of ethanol (70%, 85%, 95% I, 95% II, absolute alcohol I, and absolute alcohol II), each step lasting 3 h. The specimens were then cleared in two xylene baths (3 h each), infiltrated with molten paraffin in three successive 3-h steps, embedded in paraffin blocks, and sectioned at 6-µm thickness using a rotary microtome (HistoCore BIOCUT; Leica Biosystems, Nussloch, Germany).

Masson’s trichrome staining was employed to visualize the TCL’s cellular and extracellular matrix components. In brief, deparaffinization was carried out by immersing the sections in xylene I and xylene II for 30 min each, followed by rehydration in a graded ethanol series (absolute alcohol I, absolute alcohol II, 95%, and 80%) for 5 min per step. Slides were then rinsed in running tap water and transferred to Bouin’s fixative at 56 °C for 1 h, promoting optimal staining of collagen. After cooling and thorough washing to remove the yellow color, slides were stained with Weigert’s iron hematoxylin solution for approximately 10 min, which rendered cell nuclei a dark purple/black. They were next treated with Biebrich scarlet-acid fuchsin to stain cytoplasm and muscle fibers red. A phosphomolybdic acid-phosphotungstic acid solution was subsequently applied to enhance collagen contrast, followed by immersion in aniline blue solution, which specifically highlights collagen in shades of blue. The stained slides were quickly rinsed in distilled water, immersed in 1% acetic water for differentiation, and then dehydrated in ascending grades of ethanol (95%, absolute I, absolute II), cleared in multiple xylene baths, and mounted under a coverslip with permanent mounting media. All stained sections were analyzed under an Olympus Bx43 microscope equipped with a DP73 digital camera (Olympus Optical Co., Ltd., Tokyo, Japan) to examine collagen fiber organization, fibroblast distribution, and overall tissue architecture.

Fibroblast density was quantified by counting cell numbers per 40,000 μm² area (40X magnification field) in 60 images per age group, focusing on the youngest (<60 years) and oldest (≥80 years) groups for comparison.

Moreover, special attention was given to the connective tissue layers surrounding the nerve (mesoneurium, epineurium, perineurium, and endoneurium), as well as to the myelinated fibers and axons within the nerve fascicles.

Scanning electron microscopy

For ultrastructural analysis, a 1 × 1 cm² segment was obtained from the central region of each TCL (above the median nerve), ensuring minimal inclusion of adjacent fascial tissues. These tissue blocks were immersed in 2.5% glutaraldehyde for at least 1–2 h at room temperature to stabilize cellular and extracellular components. After initial fixation, they were rinsed twice (10 min per rinse) in 0.1 M phosphate-buffered saline (PBS, pH 7.4) to remove residual fixative, followed by three washes (10 min each) in distilled water. The samples were then post-fixed in 1% osmium tetroxide (in 0.1 M PBS, pH 7.4) for 1 h, enhancing membrane contrast and preserving ultrastructural details.

Post-fixation was followed by a graded ethanol dehydration series at 30-min intervals in 70%, 80%, 90%, 95% I, 95% II, 100% I, and 100% II ethanol. Complete dehydration was achieved by critical point drying in liquid CO₂, which prevents structural collapse of delicate collagen fibrils. Dried specimens were subsequently mounted on metal stubs using double-sided conductive tape and coated with a thin layer of gold in a sputter coater. The prepared samples were examined under a LEO 1450VP scanning electron microscope (Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 15 kV, with images acquired at low to high magnifications to visualize collagen fiber organization, interfascicular matrix distribution, and any microstructural changes within the TCL.

Statistical analysis

All biomechanical data (dynamic stiffness, logarithmic decrement, mechanical stress relaxation time, and creep) were recorded as mean ± standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA) was used to compare biomechanical properties among age groups because the independent variable (age group) was categorical and the dependent variables were continuous. Pearson’s correlation analysis was used to assess linear relationships between age and individual biomechanical parameters. One-tailed tests were applied based on a priori hypotheses that aging would be associated with increased stiffness and reduced elasticity-related properties. Correlation coefficients (r) were classified as negligible (0.00–0.10), weak (0.10–0.39), moderate (0.40–0.69), strong (0.70–0.89), or very strong (0.90–1.00) (Schober, Boer & Schwarte, 2018). Student’s t-test was used to compare fibroblast cell numbers between <60 and ≥80 age groups. A p-value less than 0.05 indicated statistical significance.

General cadaveric characteristics

Table 1 summarizes the demographic data for the 50 cadavers (100 TCLs) included in this study. The mean cadaveric age was 73.24 ± 1.25 years, with an overall age range of 40–93 years. When stratified by age groups, the mean ages were 51.14 ± 1.56 years for those under 60, 64.73 ± 0.74 for those aged 60–69, 75.40 ± 0.50 for those aged 70–79, and 85.94 ± 0.67 for those aged 80 and older. Of the total cadavers, 42% (n = 21) were male and 58% (n = 29) were female.

Mechanical property of the TCL

The TCL exhibited notable age-related changes in four key mechanical parameters: dynamic stiffness, logarithmic decrement, mechanical stress relaxation time, and creep (Table 2). Although measurements were obtained at three anatomical regions, the present analysis focused on age-related differences using averaged values rather than regional comparisons. Dynamic stiffness values in all regions—proximal, middle, and distal—rose significantly with advancing age, with a particularly large jump observed in the ≥80 age group, as indicated by the superscripts a, b, and c in Table 2 (one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference post-hoc test).

Pearson’s correlation analysis further confirmed that dynamic stiffness had moderate positive associations (r range 0.65–0.68, p < 0.001) with age across all measured locations (Table 3). In terms of elasticity, inferred from the logarithmic decrement, the highest mean values—implying the lowest elasticity—were recorded in individuals aged ≥80 years; however, only distal and average decrement values showed weak but statistically significant correlations with age (r = 0.20 and 0.21, p < 0.05). Mechanical stress relaxation time decreased markedly with age, demonstrating moderate to strong negative correlations (r range −0.67 to −0.73, p < 0.001), suggesting that stiffer TCLs require less time to rebound to their initial shape once a deforming force is removed. Finally, creep also declined sharply in older specimens, with the ≥80 group displaying significantly lower values and moderate negative correlations with age (r range −0.55 to −0.67, p < 0.001), indicative of reduced capacity for gradual elongation under sustained load. Collectively, these findings reveal that the TCL becomes progressively stiffer and less elastic with age, as evidenced by higher dynamic stiffness and logarithmic decrement, alongside lower relaxation time and creep (Fig. 1).

Histological appearance in the TCL

The TCL formed the roof of the carpal tunnel, a flat tubular structure that housed the median nerve and nine extrinsic flexor tendons. These included four tendons of the flexor digitorum superficialis, four tendons of the flexor digitorum profundus, and a single tendon of the flexor pollicis longus. Sub-synovial connective tissue was interspersed among these tendons and the median nerve. At low magnification, there were no readily noticeable differences between younger and older TCL specimens but the obvious alteration in median nerve aging was reduced connective tissue around nerve fiber (Fig. 2).

The primary structural component of the TCL was collagen fibers, which exhibited alignment in both transverse and oblique directions, contributing to the mechanical stability of the carpal tunnel. In transverse sections, collagen bundles appeared either as long, parallel structures or short, oblique segments depending on the orientation of the cut. Spindle-shaped fibroblasts were aligned along the direction of the collagen fibers, contributing to the ligament’s structural organization (Fig. 3).

In specimens from older subjects, the TCL displays pronounced age-related changes. Collagen fibers became less organized, with an increased presence of clefts between collagen bundles, indicating structural degeneration (Fig. 3B). In contrast, younger specimens exhibited well-organized collagen structures with narrow, consistent spacing between bundles (Fig. 3A). Additionally, quantitative analysis revealed a significant 41.9% reduction in fibroblast density in elderly specimens (aged ≥80 years old: 10.68 ± 0.54) compared to that in younger specimens (aged <60 years old: 18.38 ± 0.77, p = 0.009), further reflecting the age-related decline in cellularity and matrix integrity.

Scanning electron microscopic appearance in the TCL

Scanning electron microscopy revealed that TCL was covered by thin sheath tissue with a loose network of collagen fibers. The collagen fibers in the TCL were organized into tight collagen bundles. These were grouped into fascicles. The collagen bundles were irregular in shape and varied in diameter, with larger bundles predominantly located in the superficial part of the TCL and smaller bundles more common in the deep part (Fig. 4). In older specimens, there was a noticeable increase in the number of small bundles, particularly in the deep portion of the TCL (Fig. 4B), compared to younger specimens, where the bundles were larger and more consistently sized (Fig. 4A).

Additionally, fine collagen fibers were more prominently observed in the interfascicular matrix of the aging TCL. This matrix, a connective tissue surrounding the fascicles, became denser with more visible fine collagen fibers as a mesh-like pattern in the TCL in older specimens (Fig. 5B). By contrast, younger specimens displayed well-arranged collagen in parallel manner in the interfascicular matrix, resulting in a more distinct separation of fascicles (Fig. 5A). The presence of blood vessels within the interfascicular matrix was also evident and more pronounced in the young TCL.

At higher magnification, collagen fibers within the bundles were well-arranged and tightly packed in younger specimens, maintaining a regular organization (Fig. 6A). However, in older specimens, the collagen bundles were irregularly arranged and showed signs of disorganization and fragmentation, indicating significant age-related structural deterioration (Fig. 6B). These age-associated changes in the collagen arrangement and organization contribute to the stiffening and loss of flexibility in the TCL.

Histological appearance in the median nerve under TCL

The median nerve consists of multiple fascicles, each enclosing numerous axons. Each axon was surrounded by a thin connective tissue layer known as the endoneurium, while each fascicle was encapsulated by a denser connective tissue sheath called the perineurium. Surrounding all fascicles was the epineurium, a thicker sheath composed primarily of collagen fibers, which provided structural integrity to the nerve. Beyond the epineurium lay the mesoneurium, the outermost connective tissue layer that anchored the nerve to surrounding structures. The mesoneurium, epineurium, and interfascicular epineurium also contained blood vessels that supplied the nerve and its associated structures, which became progressively smaller as they penetrated deeper, transitioning into capillaries within the endoneurium (Figs. 7–9).

In aging specimens, significant structural changes were observed in these connective tissue layers. The mesoneurium and epineurium showed a reduction in thickness and collagen density in older subjects compared to younger ones (Fig. 7). This reduction narrowed the distance between the TCL and the median nerve (Fig. 7B), which may impair the nerve’s ability to expand and contract during wrist and hand movements, potentially exacerbating nerve compression under the TCL.

The interfascicular epineurium in older specimens exhibited a decrease in collagen fiber density and increased loosening, as evident in Fig. 8B. By contrast, younger specimens maintained a more compact and well-organized collagen matrix within the interfascicular epineurium (Fig. 8A). Similarly, the endoneurium surrounding individual axons showed reduced collagen density in older specimens, reflecting an age-related deterioration of this structural layer (Fig. 9B).

The myelinated fibers in the median nerve also underwent significant age-related degeneration. In younger specimens, the myelin sheaths surrounding axons were thick, continuous, and abundant (Fig. 9A). In contrast, older specimens exhibited reduced frequency and thinner, fragmented myelin sheaths. Additionally, degenerative changes were noted in the axons themselves, with evidence of axonal atrophy and a substantial loss of axons in older subjects (Fig. 9B). These findings highlight the cumulative impact of aging on the structural integrity of the median nerve, which may contribute to functional impairments and increased susceptibility to conditions such as CTS.

Clinical implications

The findings of this study have important implications for understanding the pathophysiology of CTS in older adults. The age-related stiffening and disorganization of the TCL, combined with structural degeneration of the median nerve, creates a biomechanical and neurophysiological environment that predisposes elderly individuals to CTS. This may explain why CTS is not only more prevalent in older adults but also more severe, with higher rates of functional impairment and less satisfactory outcomes following surgical intervention (Fung, Tang & Fung, 2015; Vyšata et al., 2014). Recognizing these age-related changes underscores the need for preventive and therapeutic strategies tailored to the elderly population. For example, physical therapies aimed at maintaining TCL flexibility or pharmacological approaches targeting MMP activity and glycation may help mitigate these structural changes.

Study limitations and future directions

This study is not without limitations. The use of formalin-embalmed cadavers may alter the mechanical properties of the TCL, potentially affecting the generalizability of the findings to living tissues. This study did not evaluate the role of proteoglycans or ground substances, which are integral components of the extracellular matrix and may significantly influence ligament mechanics. Additionally, the absence of detailed medical histories may represent a potential confounding factor, as systemic diseases could influence ligament and nerve properties. This study focused on proximal–distal variations of the TCL overlying the median nerve; mediolateral thickness and detailed nerve pathway orientation were not evaluated and warrant further investigation. The unequal distribution of specimens across age groups, particularly the smaller number of younger cadavers, reflects the inherent nature of cadaveric studies and may introduce bias favoring older age groups. Future research also should investigate these components and explore therapeutic interventions that could slow or reverse age-related changes in the TCL and median nerve. Furthermore, in vivo studies using advanced imaging and biomechanical modeling could provide a more comprehensive understanding of carpal tunnel biomechanics in aging populations.