Studies on the Innervation of the Medial Meniscus in the Human Knee Joint' A. S. WILSON,2 P. G. LEGG3 AND J. C. McNEUR Medical School, Monash University, Clayton, Victoria, Australia ABSTRACT Histological and ultrastructural studies on the medial meniscus in the human knee joint show that nerve fibres, both myelinated and unmyelinated, extend from the periarticular plexus into the meniscus as far as the intermediate third. These neural elements are not exclusively paravascular in position and it is reasonable to postulate a function other than vasomotor or vasosensory for them. The topographical features of the innervation of the adult human knee joint, as described by Schaeffer ('42) and Hollinshead ('66) are well understood but the precise extent to which nerve fibres penetrate intra-articular structures such as the menisci remains undefined. The functional studies of Samuel ('52) and Andrew ('54) indicating that neural elements are present within periarticular structures agree with the histological findings of Rossi ('50) and Polacek ('61) but the question of meniscal innervation is not considered. Gardner ('48) in an extensive histological survey, reports that, in foetal human knee joints, some vessels and nerves enter the substance of the menisci but no exact value is given for the depth of their penetration. Freeman and Wyke ('67) state that those few nerve fibres present within menisci of adult cats are confined to the outermost layers, occupy paravascular positions and possess no specialized endformations. They report that no neural elements can be seen within the central fibrocartilaginous zone. In the histological part of this investigation, an attempt is made to determine more precisely, the extent and nature of the nerve fibres which ramify within adult human menisci. Accurately orientated portions of menisci are subjected to electron microscopy so that comparisons may be made between the histological and ultrastructural features of meniscal innervation. Medial menisci were obtained at operation from 10 male patients, aged 16 to 29 years, who had suffered traumatic knee injury. Only menisci in which the damage ANAT. REC., 165: 485-492. was clearly localized to either the anterior or posterior horn were used. ( a ) Histological studies : Immediately on removal, eight menisci were fixed in 10% formalin for approximately 12 hours, after which both anterior and posterior horns were removed and discarded, thus leaving macroscopically undamaged middle portions as indicated by B (fig. 1 ) . These portions were then placed in 10% formalin over marble chips for a further 10 days. Three portions were embedded in paraffin wax, and serial sections, representing three distinct planes were cut at a thickness of 5 v, and stained, either with haematoxylin and eosin or by Mallory's trichrome procedure. From the remaining five portions, frozen sections were cut at a thickness of 100 and impregnated with silver according to Schofield ('60). (b) Ultrastructural studies: Three specimens, each approximately 3 mm in thickness, and orientated as shown (fig. 2) were cut at operation from the middle portion of two menisci. All peripheral connective tissue was removed and they were then immersed in phosphate buffered 4% distilled glutaraldehyde for one hour at 4°C. Each specimen was then divided longitudinally into two narrow strips and fixed for a further three hours in glutaraldehyde. Postfixation was carried out in ice-cold 2.5% osmium tetroxide buffered with a potassium dichromate-calcium chloride mixture (Richardson, '62). After dehydration and Received Dec. 2, '68. Accepted June 18, '69. 1 A preliminary report on these studies was presented at the Annual Meeting of the Anatomical Society of Australia and New Zealand in May 1968. 2 Present address: Department of Anatomy, University of Western Ontario, London, Canada. 3 Present address: Department of Anatomy, University of Minnesota, Minneapolis, Minnesota. 485 486 A. S. WILSON, P. G. LEGG embedding in Araldite, thin sections were cut, stained with lead citrate (Reynolds, '63) and examined under the electron microscope. During these procedures, care was taken to ensure accurate orientation. For purposes of description, the portions of meniscus, orientated as 2 and 3 (fig. 2) were divided into internal, intermediate and external thirds by measurement (fig. 3). Sections were cut in sequence from internal to external zones until neural tissue was positively identified. OBSERVATIONS ( a ) Histological studies : In sections cut at right angles to the long axis of the limb and stained with haematoxylin and eosin or Mallory's trichrome stain, conspicuous indentations, containing lightly stained connective tissue were evident at irregular intervals along the peripheral border of the meniscus. This finding was confirmed in specimens which represented other planes of section. Vascular structures within these indentations were generally directed at right angles to the margin of the meniscus (fig. 4). In silver preparations, periarticular nerve fibres were traced to the external border of the meniscus where the majority appeared to terminate, although no specialized end-formations were observed. However, on approaching the meniscus, some fibres changed their general direction from radial to circumferential. These comprised small myelinated fibres approximately 5 CI in diameter and fasciculi of unmyelinated fibres (fig. 5). Some of these nerve fibres penetrated the external border of the meniscus at the regions of indentation formed by the capsular fibrous tissue apd although some were clearly paravascular, others arborized at some distance from vessels within the external third. .On several occasions neurovascular bundles were identified within small interstitial areas of connective tissue, usually near the junction between external and intermediate thirds (fig. 6). Occasionally, tortuous argyrophilic axis cylinders were traced from the periarticular tissue through the external third and deep into the intermediate third of the meniscus where capillary loops were also seen (fig. 7). AND J. C. 1McNEUR The predominant cell type in the external third was elongated and resembled a fibroblast whereas, at the internal margin of the meniscus, the majority of cells were round to ovoid in outline. These were commonly seen in pairs and resembled cartilage cells. Cells of the intermediate third portrayed some features of both of these distinct types (fig. 8). ( b ) Ultrastructural studies: In all of the specimens examined, fasciculi of nerve fibres cut in various planes, were present in the external third of the meniscus. The largest fasciculi generally contained 1-4 myelinated fibres which ranged in diameter from 1.5-4 p and 30-50 unmyelinated fibres. Each large fasciculus was surrounded by a distinct perineurial sheath (fig. 9 ) . The smallest fasciculi contained only unmyelinated fibres which were characteristically devoid of perineurium, but which were accompanied by a co-axial covering of collagen fibres (fig. 10). While it was obvious that some fasciculi were paravascular in position, many were embedded in the matrix at some distance from vessels. Nerve fibres were also clearly identified in the intermediate third of the meniscus. Most of these comprised small groups of unmyelinated fibres enveloped in Schwann cell coverings and closely resembled the smallest fasciculi in the external third. NO specialized end-formations were observed. Within the internal third of the meniscus no neural elements were identified with certainty. Throughout the intermediate third, the fine structure of some cells resembled that of cartilage cells. However, in the internal third, the resemblance of the predominant Fig. 1 Indlicates the position of portion B which was used for histological and ultrastructural studies. Portion A , anterior horn and B, posterior horn, were discarded. Fig. 2 Illustrates the relationship of specimens used for ultrastructural studies to the CIOSSsection of the meniscus. Fig. 3 The broken lines indicate the boundaries between external, intermediate and internal thirds of the meniscus. Fig. 4 Part of a medial meniscus stained with haematoxylin and eosin. A branch ( B ) from a periarticular vessel can be seen approaching the external margin of the meniscus through an indentation of pale-staining capsula tissue. INNERVATION OF MEDIAL MENISCUS Figures 1-4 487 488 A. S. WILSON, P. G. LEGG connective tissue cells to cartilage cells was most prominent (fig. 11). DISCUSSION AND J. C. McNEUR specialized insulation which might be expected in this potentially stressful situation. The absence of perineurial coverings from the small fasciculi suggests that the axis cylinders may come into closer contact with adjacent meniscal cells but further investigations will be required to examine this possibility. The morphological differences in predominant connective tissue cell types within external, intermediate and internal thirds of the meniscus are conspicuous in both histological and ultrastructural preparations. The cell population is predominantly fibroblastic in the external third but is chondrocytic in the internal third. The cells of the intermediate third of the meniscus can not be placed definitely in either of these categories and it seems probable that they are intermediate, not only in position, but also in cell type. It is not possible to ascribe definite function to the intrameniscal nerve fibres, but the most important structural considerations are tlhe relative sparsity of nerve fibres, their comparatively small diameter and the absence of demonstrable specialized end-formations. Excluding paravascu- The ease with which the free internal border of the medial meniscus can be recognized contrasts sharply with the difficulty in demarkating the attached external border, the position of which is essential for accurate orientation of nerve fibres within the meniscus, In this investigation, the outermost layer of circumferentially-directed connective tissue is considered to be the external limit of the meniscus and it seems probable that this outermost layer is equivalent to the “marginal zone” of Schaeffer (’42) and to the “annular ligament” of Freeman and Wyke (’67). Silver studies confirm the observation in haematoxylin and eosin preparations and in sections prepared by Mallory’s method that the external boundary of the meniscus is indented at irregular intervals by pale-staining connective tissue from the capsule. These indentations contain neurovascular bundles and thus establish portals of entry into the meniscus for both vessels and nerve fibres. The continuity, observed on several occasions between periarticular 5 Silver preparation showing a fasciculus and intrameniscal nerve fibres, clearly indi- of Fig the periarticular plexus (PP). It is approxicates that the meniscal nerve fibres are de- mately parallel to the external margin of the rived from the periarticular plexus. More meniscus which is indicated by the arrow. Fig. 6 Illustrates a n interstitial area near the frequently, however, axis cylinders were seen either as short lengths, directed radi- junction between external (E) and intermediate ( I ) thirds of the meniscus. One vascular strucally, or as components of neurovascular ture can be identified and the argyrophilic strucbundles which occupied interstitial areas tures ( A S ) indicate nerve fibres cut transversely. within the meniscus. The paucity of deFig. 7 Tortuous nerve fibre (NF) which was monstrable long lengths of fibres may be traced from th(e periarticular tissue into the interthird o f the meniscus. Note the rounded attributable to tortuosity of the axis cyl- mediate profiles of adjacent connective tissue cells. inders. Thus histological studies show that Fig 8 Samples of cells present in (1) external, the innervation of the meniscus is more ex- ( 2 ) intermediate and (3) internal thirds of the tensive than previously reported, but they meniscus. Fig. 9 The largest fasciculus of nerve fibres provide no detailed information on the intercellular relationships of neural compon- seen. This was situated close to the junction of external and intermediate thirds of the meniscus. ents. Perineurium (P), myelinated fibres (MYF) and Ultrastructural studies clearly demon- Schwann cell nuclei (SCN) are clearly evident strate that nerve fibres are present within along with many unmyelinated fibres. Fig. 10 A small fasciculus of unmyelinated the external and intermediate thirds of the nerve fibres (1JMF) within the external third of meniscus. The largest fasciculi which con- the meniscus. The blood vessel closest to these tain a few myelinated fibres of small diam- fibres was on the other side of a connective tissue eter, Schwann cells and collagen fibres and cell (CTC). Fig. 11 Illustrates a cartilage cell which was which are surrounded by a perineurial present in the internal third of the meniscus. sheath, closely resemble peripheral nerves Villous projections (VP), apparently vacant peridescribed by Gamble and Eames (’64) cellular spaces ( S ) and surrounding pseudomemand Wilson (’65). There is no evidence of brane (MEM) are conspicuous features. Figures 5-8 Figures 9-11 INNERVATION OF MEDIAL MENISCUS lar components which are probably vasomotor or vasosensory, these features suggest that intrameniscal fibres may perform an afferent function characterized by a relatively low conduction velocity. It seems most likely that the modality which they transmit is “slow” pain. LITERATURE CITED Andrew, B. L. 1954 The sensory innervation of the medial ligament of the knee joint. J. Physiol. (Lond.), 123: 241-250. Freeman, M. A. R., and B. Wyke 1967 The innervation of the knee joint. An anatomical and histological study i n the cat. J. Anat. (Lond.), 101: 505-532. Gamble, H. J., and R. A. Eames 1964 An electron microscope study of the connective tissues of human peripheral nerve. J. Anat. (Lond.), 98: 655-663. Gardner, E. D. 1948 The innervation of the knee joint. Anat. Rec., 101: 109-130. Hollinshead, W. H. 1966 In Anatomy for Surgeons, pp. 786787. Hoeber-Harper International Edition, London and Tokyo; Harper and Row. 49 1 PolPcek, P. 1961 Differences in the structure and variability of encapsulated nerve endings in the joints of some species of mammals. Acta. Anat., 47: 112-124. Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron-opaque stain in electron microscopy. 3. Cell. Biol., 17:207. Richardson, K. C. 1962 The fine structure of autonomic nerve endings in smooth muscle of the r a t vas deferens. J. Anat. (Lond.), 96: 427442. Rossi, F. 1950 Sur l’innervation fine de la capsule articulaire. Acta Anat., 10: 161-232. Samuel, E.P. 1952 The autonomic and somatic innervation of the articular capsule. Anat. Rec., 113: 53-70. Schaeffer, 3. P. 1942 In Morris’ Human Anatomy, p. 273, Edition 11, Blakiston, New York and Toronto. Schofield, G. C. 1960 Experimental studies on the innervation of the mucous membrane of the gut. Brain, 83: 490-514. Wilson, A. S. 1965 Electron microscope studies on the nerve fibres and vessels of the phrenic nerve. In: Proceeding of the Eighth International Congress of Anatomists, Wiesbaden, 1965.