Decrease in the numbers of mechanoreceptors in rabbit ACL: the effects of ageing

  • Sedat Tolga Aydoğ
  • Petek Korkusuz
  • Mahmut Nedim Doral
  • Onur Tetik
  • Haydar Ali Demirel
Knee

Abstract

It is proposed that there is a positive correlation between the numbers of mechanoreceptors and proprioceptive function. On the other hand, normal aging process is associated with deficits in proprioception. This study is designed to test the hypothesis that aging resulted in decreased numbers of mechanoreceptors. Anterior cruciate ligaments of 14 male rabbits (2 months, n=5; 12 months, n=4 and 60 months, n=5) were extracted and the total numbers of Ruffini, Pacini and Golgi tendon-like receptors were accounted. As a result, the numbers of mechanoreceptors, especially Ruffini receptors, decreased with aging (p<0.05). Increased age was associated with changes in the morphology of mechanoreceptors. In conclusion, aging results in both diminished numbers and changed morphology of mechanoreceptors.

Keywords

Ageing Proprioception Mechanoreceptor Light microscope ACL 

Introduction

Proprioceptive function has been shown to decline with increasing age for both sexes [1, 2, 3, 4, 5]. This age-related decrement might be result from the loss of mechanoreceptors, altered muscle function or the consequent joint instability, spinal cord reflexes and impairment of central nerves system [3]. Mechanoreceptors (MRs) are of special interest in this study, since there is a positive correlation between the number of MRs and accuracy of the joint position sense [6].

Most investigators consider ligaments to be a passive stabilizer of the joints. However, for more than a century, investigators have recognized the presence of MRs in the ligaments and potential roles of the ligaments in proprioception [7]. To the best of our knowledge, there is only one study that showed decline in the numbers of MRs in ligaments with ageing [8]. In that study, subjects consisted of both patients with chronic shoulder problems and acute shoulder fracture. Since, untreated ligament lesions in humans resulted in decreases and eventually total loss of the numbers of mechanoreceptors [9], there is a need for additional study designed with healthy subjects to clarify the effects of aging on MRs.

The aim of this study was to investigate the effects of aging on the numbers and the morphological changes of the MRs in the anterior cruciate ligament (ACL). We have hypothesized that aging resulted in decreased numbers of mechanoreceptors.

Materials and methods

Animal model

Fifteen New Zealand rabbits were classified into three groups; 2 months (young; n=5), 12 months (adult; n=5), and 60 months (old; n=5). Since there were osteoarthritic changes in the knee of one rabbit from adult group, this rabbit was excluded from the study and 14 rabbits were used for histological analysis. The Ethical Committee of the University approved all procedures (17/2/2003, 04/01/2003). Surgical procedures were carried on right knees of all of the rabbits. ACLs from right sides were obtained by cutting both tibial and femoral attachments.

Histological analysis

All specimens were evaluated by histologist who did not know the study groups. The histological method was modified from Adachi et al. [6] and Zimny et al. [10]. The retrieved ACL specimens were fixed in 10% phosphate buffered formalin (pH 7.0) at room temperature, rinsed in the buffer, and decalcified in 88% formic acid and lemon juice (3:1) solution in a dark room for 15 min. They were immersed in 1% gold chloride solution in a dark room for 20 min, for block staining. The specimens stayed in 25% formic acid in a dark room for 15 h. The specimens were then washed in running tap water for 1 h, flash frozen in liquid nitrogen at −196°C and stored in this condition till sectioning. Thick (60 μm) longitudinal, serial sections were prepared with a cryo-microtome (Leica, Germany). The sections were mounted on gelatin-coated slides.

All serial sections were examined under a light microscope (Leica, Germany) by at least two investigators using the morphological criteria of Freeman and Wyke [11] for the presence of the MRs. The images were captured via Leica DC500 digital camera.

The mechanoreceptors were classified as Ruffini receptors, Pacini receptors, Golgi tendon-like receptors, and free nerve endings. The total numbers of nerve endings were excluded from the count, since there were so many free nerve endings, which may interfere with the results.

Statistical analysis

The total histological scores were expressed as means and standard deviations. The data was analyzed using non-parametric Mann Whitney U test and the Kruskal Wallis test to assess statistical significance. Probability value of less than 0.05 was considered as significant.

Results

Mechanoreceptors were distinguished in all of the specimens according to the morphological criteria from Hasegawa et al. [12]. Both the vascular structures and MRs were stained by gold chloride in the connective tissue septa. The mechanoreceptors were frequently observed in close contact with the vascular structures. The numbers of mechanoreceptors for each MR groups and mean of each groups are presented in Table 1.
Table 1

The numbers of mechanoreceptors for each MR groups and mean of each group

Group

Type 1 Ruffini

Mean

Type 2 Pacini

Mean

Type 3 Golgi

Mean

Total MRs

Young

15, 13, 10, 19, 11

13.6 ± 3.58**

8, 6, 9, 10, 6

7.80 ± 1.79*

3, 4, 4, 4, 2

3.4 ± 0.89*

8.27 ± 4.85a

Adult

6, 8, 9, 8

7.75 ± 1.26**

8, 4, 4, 9

6.25 ± 2.63

3, 2, 2, 3

2.5 ± 0.58

5.50 ± 2.78b

Old

5, 7, 8, 5, 2

5.40 ± 2.30**

2, 4, 5, 4, 2

3.40 ± 1.34*

2, 1, 1, 2, 2

1.6 ± 0.55*

3.47 ± 2.17c

*The difference between young and old groups is statistically significant (p<0.01)

**The difference between young and other two groups is statistically significant (p<0.01). No difference between adult and old groups

aThe difference between young and adult groups is statistically significant (p<0.05)

bThe difference between young and old groups is statistically significant (p<0.01)

cThe difference between adult and old groups is statistically significant (p<0.05)

Except the free nerve endings, most of the MRs were observed in close proximity to bony insertions of the ACLs. On the other hand, free nerve endings were rather homogenously distributed throughout the ligaments (Fig. 1). The receptors were rarely detected in the deep part of the ACLs. Some Pacini receptors, Ruffini receptors and Golgi tendon organs that had irregular margins or flattened configurations were noted especially in the old group (Fig. 2). Although no measurements and statistical analyses were performed for the size of MRs, all receptors of the young group were relatively smaller in size compared to other two groups.
Fig. 1

a A dendritic Ruffini receptor from the young group specimen (Gold chloride, 400×). b A small Golgi tendon organ-like receptor from the young group specimen (Gold Chloride, 400×). c A Pacini corpuscle with its lamellary configuration from the young group specimen (Gold Chloride, 400×). d A panoramic view of the frequently distributed free nerve endings in young groups ACL (Gold Chloride, 200×). e A Ruffini receptor with its relatively tiny processes comparing to micrograph. a Adult ACL (Gold Chloride, 400×). f A Golgi tendon organ-like receptor from an adult ACL(Gold Chloride, 400×)

Fig. 2

a, c The modified Golgi tendon-like receptors in old ACL (Gold Chloride, 400×). b, d The modified Pacini receptors in old ACL (Gold Chloride, 400×)

Golgi tendon organ-like receptors were the rarest MRs in all groups. The numbers of Ruffini, Pacini and Golgi tendon organ-like receptors decreased significantly in the old group compared to the young group (p<0.05). Besides, the number of Ruffini receptors of adult ACLs was significantly lower than that of the young group (p<0.05). The total numbers of MRs were significantly decreased with increasing age and the differences were statistically significant between young and other two groups (p<0.05). Though the exact number is not counted, the numbers of the free nerve endings were apparently higher in the young group compared to other groups. A slight variation was observed regarding the size of the receptors between the young and other two groups.

Discussion

In this study, we have investigated the effects of aging on the numbers of the MRs in ACLs of healthy rabbits. We have also examined the age-related changes in the morphological properties of the MRs. Our results have clearly demonstrated that aging was associated with decreased numbers of mechanoreceptors. Brief discussions of our findings are following.

Proprioceptive capacity is influenced by muscle and joint function, mechanoreceptors, spinal cord reflexes, and brain. Proprioceptive function has been shown to decline with increasing age for both sexes [1, 2, 3, 4, 5]. Indeed, Salo and Tatton [13] have provided the data about the age-related loss of dorsal root ganglions innervating knee joint in ageing mice. It is also well known that muscle strength and nerve conduction velocity decline with increasing age [3, 14]. On the other hand, there is insufficient data regarding the effects of aging on MRs. MRs are special structures to sense and transmit the signals to the central nervous system about the joint angle, mechanical stress, acceleration and deceleration of the movement [8, 10, 11, 15]. Adachi et al. [6] showed positive correlation between the number of MRs and the joint position sense, which is the most common test to evaluate proprioceptive functioning. Therefore, age-associated decrease in proprioceptive functioning might be related to the reduction in the numbers of MRs. To the best of our knowledge, there is only one study showing the decline in the number of the MRs with ageing [8]. In that study, authors evaluated the coracoacromial ligament obtained during the operation of scapula and humeral neck fractures or chronic impingement syndromes. Since decreased numbers of MRs in the ruptured ACLs have been reported [9], any impairment in the components around joint might result in decline in the numbers of MRs independent of age. Therefore, Morisawa’s findings, especially for specimens obtained from the cases with impingement syndrome, should be reevaluated for chronic changes of MRs caused by the pathologic conditions. Nevertheless, our study has clearly indicated that increasing age was associated with decreasing numbers of MRs in healthy rabbits. This age-related decline in the MRs were more pronounced in the Ruffini receptors since both old and adult rabbits represent significantly lower numbers of receptors than that of young animals. Furthermore, there was a stepwise decrease in the distribution of the total numbers of MRs as a function of age (young>adult>old).

In our study, similar to the Freeman’s findings [7, 11], ACLs are primarily composed of Ruffini receptors and free nerve-endings. Golgi tendon organ-like receptors were the lowest numbers of receptors found in all groups in our study. Some modified forms of Pacini and Ruffini receptors and Golgi tendon organs have been noted with either irregular margins or flattened configurations especially in the old group. All receptors of the young group were relatively smaller in size compared to the other two groups. Although we do not know the physiological meaning of this age-related changes in the morphology of MRs, this could be an exciting area for future research.

In our study, there were no changes in the localization of the MRs with aging. Except free nerve endings, MRs were very rarely detected in the deep part of the ACL and higher numbers of them have been observed in close proximity to bony insertions. On the other hand, similar to other findings [11, 15, 16], free nerve endings were distributed throughout the ligaments. It is known that the MRs located in close proximity to the origin or insertions of the ligaments simply perceive loads of pressure or tension [8, 10, 11, 16]. Therefore, age-associated reduction in the numbers of MRs, which are especially located in that area might indicate the loss of load bearing capacity of elderly people.

Nonetheless, decreased numbers of the MRs with aging found in our study may suggest that increasing age may result in impaired propriocepive functioning. Impaired proprioception may be a contributing factor to the falls which are known to lead to significant morbidity and mortality in people older than 65 years. It has been reported that balance performance for both sexes changes with chronological age and a progressive decline was seen after the third–fourth decade of life [1]. Furthermore, some authors showed that regular physical activity might attenuate the age-related decline in the proprioceptive functioning [1, 4, 17]. Further studies are needed to show if habitual physical activity attenuates age related decline in the numbers of MRs.

In conclusion, this study for the first time, has shown that the numbers of MRs decrease with age in the ACL of healthy subjects. Increased age was also associated with the changes in the morphology of the mechanoreceptors.

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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Sedat Tolga Aydoğ
    • 1
  • Petek Korkusuz
    • 2
  • Mahmut Nedim Doral
    • 1
    • 3
  • Onur Tetik
    • 1
  • Haydar Ali Demirel
    • 1
  1. 1.Department of Sports Medicine, Faculty of MedicineHacettepe UniversityAnkaraTurkey
  2. 2.Department of Histology and Embryology, Faculty of MedicineHacettepe UniversityAnkaraTurkey
  3. 3.Department of Orthopedics, Faculty of MedicineHacettepe UniversityAnkaraTurkey

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