The Veterinary Journal 205 (2015) 413–416
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Short Communication
Sympathetic innervation of the suprasesamoidean region of the deep digital flexor tendon in the forelimbs of horses F. Beccati *, M. Pepe, L. Pascucci, P. Ceccarelli, E. Chiaradia, F. Mancini, M.T. Mandara Centro di Studi del Cavallo Sportivo, Dipartimento di Medicina Veterinaria, Università degli Studi di Perugia, Via San Costanzo 4, 06126 Perugia, Italy
A R T I C L E
I N F O
Article history: Accepted 4 June 2015 Keywords: Horse Deep digital flexor tendon Tyrosine hydroxylase Alpha-1 adrenergic receptor Sympathetic innervation
A B S T R A C T
The purpose of this study was to delineate the pattern of sympathetic innervation in the suprasesamoidean region of the deep digital flexor tendon (DDFT) in horses using immunohistochemical staining (IHC) for tyrosine hydroxylase (TH) and alpha-1 adrenergic receptor (α1-AR). Fourteen forelimbs were collected from 10 horses. Longitudinal sections of the suprasesamoidean region of healthy DDFTs were harvested. Most of the sympathetic innervation was found to be in the walls of blood vessels. The tendon tissue proper was sparsely innervated, with a lesser degree of innervation within the dorsal fibrocartilage. Increased α1-AR immunostaining was also detected in walls of blood vessels and in spindle cells of fibrocartilage. Both α1-AR and TH immunostaining were detected in tenocytes. These findings support the presence of autocrine/paracrine catecholaminergic signalling in equine tendon tissue. © 2015 Elsevier Ltd. All rights reserved.
Progress in the field of diagnostic imaging has shown that the deep digital flexor tendon (DDFT) (M. flexor digitalis profundus) is one of the most commonly injured structures in sport horses undergoing magnetic resonance imaging for foot pain, with a total prevalence ranging from 30% to 83% (Schramme, 2011). The macroscopic appearance of the tendon as well as its histological structure and various pathohistological lesions has been previously described (Blunden et al., 2006). Blunden et al. (2006) suggested that DDFT lesions have a degenerative rather than inflammatory pathogenesis. The peripheral nervous system participates in the regulation of cell proliferation, release of cytokines and growth factors, modulation of inflammation and immune responses, and hormone release (Ackermann, 2013). It has, therefore, been proposed that abnormalities in neurotransmitter function might be related to the degenerative events in tendinosis in humans (Scott and Bahr, 2009). The nerves innervating tendons are mostly composed of nonmyelinated, slow-transmitting Aγ-, Aδ-, B-, and C-fibres with a small fraction of myelinated, fast-transmitting Aα- and Aβ-fibres. Mechanoreception, nociception, and vasomotor modulation are three primary functions of tendon innervation (Ackermann, 2013). Autonomic innervation is involved in the dynamic regulation of tendon blood flow and includes parasympathetic and sympathetic components that mediate vasodilatation and vasocostriction, respectively (Scott and Bahr, 2009). Tyrosine hydroxylate (TH) is the rate-limiting enzyme in sympathetic neuropeptide synthesis and alpha-adrenergic
* Corresponding author. Tel.: +39 075 585 7713. E-mail address: [email protected] (F. Beccati). http://dx.doi.org/10.1016/j.tvjl.2015.06.004 1090-0233/© 2015 Elsevier Ltd. All rights reserved.
receptors (α1-AR) are the main autonomic receptors expressed in tendon (Scott and Bahr, 2009) thus representing the main biomarkers for sympathetic innervation in tendons. Although tendons have traditionally been considered as hypoinnervated, data regarding the innervation pattern of the DDFT in horses are scant. The aim of this study was to determine the pattern of sympathetic innervation in the suprasesamoidean region of the normal equine DDFT in the forelimb using immunohistochemistry (IHC) and TH and the α1-AR as markers of different functional phases of the same autonomic system. Fourteen feet were collected from 10 horses with no history of forelimb-foot pain that had been humanely euthanased for reasons not related to this study. Signalment details of horses are presented in Table 1. Post-mortem, radiographic and ultrasonographic examinations of the feet were performed to verify that the DDFT was of normal appearance and as previously described (Bolen et al., 2007; Butler et al., 2008). To exclude the presence of tendon lesions, gross and histological examinations of the DDFT were also performed. All procedures were approved by the Institutional Animal Care Committee of the University of Perugia. Tissue samples were collected immediately after death and the suprasesamoidean region of the DDFT was excised. For each DDFT, three full-thickness samples were selected for the histological and IHC examinations (Fig. 1). Samples were fixed in 10% neutralbuffered formalin, dehydrated and embedded in paraffin wax; 4 μm thick sections were stained with Harris’s haematoxylin and eosin, Masson’s trichrome stain, aldehyde fuchsin, and Verhoeff’s stain to characterise tissue morphology. Immunohistochemical staining was performed using the 1B5 mouse anti-human tyrosine hydroxylase monoclonal antibody (TH; 1:40; Leica-Novocastra) and a rabbit antihuman α1-AR polyclonal antibody (1:80; antibodies-online).
414
F. Beccati et al./The Veterinary Journal 205 (2015) 413–416
Table 1 Summary of the signalment details of the animals used in this study.
1 2 3 4 5 6 7 8 9 10
Breed
Age
Sex
Previous use
Warmblood Warmblood Anglo-Arab Anglo-Arab Arab Warmblood Anglo-Arab Pony Thoroughbred Thoroughbred
14 21 15 22 16 13 23 21 7 3
Female Gender Female Female Gender Gender Gender Gender Female Female
Show jumping Show jumping Show jumping General purpose Endurance Show jumping Endurance General purpose General purpose Race
tendon, consisting of dense connective tissue with fibres oriented in a longitudinal fashion and rich in blood vessels. The anti-human α1-AR antibodies were validated for detection of the equine α1-AR protein in the IHC by Western blotting (WB) according to Bordeaux et al. (2010). Briefly, protein samples, obtained from the equine liver (20 μg), brain (20 μg), and tendon (40 μg), were separated by 12% polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred to nitrocellulose membranes, probed with anti-human α1-AR antibody (1:500) and detected with horseradish peroxidase conjugated goat anti-rabbit IgG polyclonal antibody (Santa Cruz Biotechnology). The immunoreactive bands were visualised with ECL reagents and by exposing X-ray films. The WB analysis (Fig. 2) indicated that the anti-human α1-AR antibody used is specific for the detection of equine α1-AR protein because a single band corresponding to approximately the expected molecular weight of the α1-AR (~50 kDa) protein was detected in the liver, brain and tendon line. Further, no extraneous bands were detected (Bordeaux et al., 2010). Based on previous studies in equine nervous tissue, WB validation for TH was not performed (Russo et al., 2010). Zone 1 was scantly vascularised, except for the superficial subsynovial stratum. The distal region of this layer contained collagen fibres that ran at angles to one another and numerous isolated or grouped chondrocytes (Fig. 3a). No TH immunoreactivity was observed (Fig. 3d) while a remarkable cytoplasmic immunoreactivity for α1-AR was detected in scattered spindle cells (Fig. 3g). Zone 2 was characterised by an extensive inter-fascicular vascular network that extended throughout the loose connective tissue of the endotendon (Fig. 3b). Tenocytes were the most common type of cells and were arranged in long parallel rows among collagen fibres. Slight to remarkable TH-immunoreactivity was detected, consisting of occasional positive nerve fascicules and tenocytes (not
Fig. 1. Diagram to show the sites (black dotted lines) for collection of samples of the suprasesamoidean region (white dotted lines) of the deep digital flexor tendon for histological and immunohistochemical examinations. SNC, synovium of the navicular bursa.
After deparaffinisation and rehydration, the endogenous peroxidase was inactivated by incubating the sections in 3% hydrogen peroxide (for TH) or methanol (for α1-AR) at room temperature. The slides were probed with the primary antibodies overnight at 4 °C. Antigen retrieval was not performed. Immunoreactivity was detected using an avidin–biotin method (LSAB+/System-HRP, DakoCytomation) and aminoethyl carbazole as the substrate (AEC + Substrate-Chromogen Ready-to-use, DakoCytomation). Carazzi’s haematoxylin was used as a counterstain. Paramount mounting medium (DakoCytomation) was used to mount the coverslips on the slides. For each tendon sample, immunolabelling staining intensity was assessed as one of three grades (absent, slight and remarkable). The equine midbrain was used as a positive control in the IHC experiments, as indicated by the manufacturers. Negative controls were carried out in the same manner with omission of the primary antibody. For the histomorphological and IHC analyses, the DDFT sections were divided into three different zones according to Blunden et al. (2006): (1) zone 1, the most dorsal layer of the DDFT (approximately 30% of the entire tendon thickness) consisting of dense irregular connective tissue and rich in elastic fibres; (2) zone 2, primarily dense regular connective tissue, exhibiting closely packed longitudinal collagen bundles; (3) zone 3, the palmar layer of the
Fig. 2. Validation of α1-AR antibody for equine proteins. Total protein extract from equine liver (20 μg), brain (20 μg) and tendon (40 μg) were analysed by Western blotting to test the specificity of the antibody. A single band corresponding to approximately the expected molecular weight of the α1-AR (~50 kDa) protein was detected in each line. No extraneous bands were detected.
F. Beccati et al./The Veterinary Journal 205 (2015) 413–416
415
a
b
c
d
e
f
g
h
i
Fig. 3. Equine deep digital flexor tendon (DDFT). Normal morphology (a–c) and sympathetic system (d–i). (a) Zone 1. Fibrocartilaginous tissue with well differentiated nest of chondrocytes (Masson’s trichrome stain, ×20); (b) Zone 2. Dense regular connective tissue rich in tenocytes and endotendon fibrovascular septa (H&E, ×20); (c) Zone 3. Blood vessels of different diameter embedded in abundant connective tissue (Masson’s trichrome stain, ×20); (d) Zone 1. No elements of this zone are positive for TH (ABCmethod and Carazzi’s haematoxylin, ×20); (e) Zone 2. Marked TH-immunoreaction showed by small blood vessel of endotendon septa (ABC-method and Carazzi’s haematoxylin, ×20); (f) Zone 3. The TH-immunoreaction markedly occurs in media–adventitia layers of blood vessels (ABC-method and Carazzi’s haematoxylin, ×20); (g) Zone 1. Scattered spindle cells show a marked cytoplasmic α1-AR immunoreaction (ABC-method and Carazzi’s haematoxylin, ×20); (h) Zone 2. Marked α1-AR immunoreaction expressed by tenocytes (ABC-method and Carazzi’s haematoxylin, ×20); (i) Zone 3. Cellular elements of vascular media–adventitia layers express a marked α1-AR immunoreaction (ABCmethod and Carazzi’s haematoxylin, ×20).
shown); in two cases, a remarkable level of TH-immunoreactivity was associated with the blood vessels of the endotendon septa (Fig. 3e). Foci of remarkable immunoreactivity for α1-AR were sparsely distributed in the walls of the blood vessels in the endotendon septa and in the tenocytes (Fig. 3h). In the tendon proper tissue, positive nerve fibres showed remarkable immunoreactivity for α1-AR. Zone 3 was rich in blood vessels (Fig. 3c). The IHC staining for TH ranged from slight to remarkable. The TH immunoreactivity pattern indicated the presence of nerve profile in the proximity of blood vessels, and in a large extent located at media–adventitia borders (Fig. 3f). The IHC staining for α1-AR was slight to remarkable in the smooth muscle cells of the tunica media of the arterioles (Fig. 3i). The results imply a number of interpretations. Firstly, TH immunoreactivity associated with the blood vessels, particularly as observed in zone 3, is consistent with previous studies of TH in the patellar tendon (Danielson et al., 2007) and the Achilles tendon (Bjur et al., 2008) in humans. Secondly, the sympathetic innervation
detected in the blood vessels and nerve fascicules in zone 2 was less extensive than that observed in zone 3, although THimmunoreactivity was occasionally observed in the tenocytes; recent studies in humans have shown that tendon neurotransmitters, including catecholamines, may be produced by tenocytes (Scott and Bahr, 2009). Thirdly, the complete absence of TH-immunoreactivity in zone 1 is consistent with findings observed in previous human studies (Bjur et al., 2008), which showed that the level of innervation decreased with increasing distance from the loose connective tissue. Fourthly, the pattern of sympathetic innervation determined in IHC analysis for the TH was confirmed in the IHC analysis of α1-AR immunoreactivity in the tunica media of arterioles scattered throughout zone 3 and in the walls of blood vessels in zone 2, further indicating the presence of sympathetic nerve fibres. However, the α1-AR immunoreactivity in zone 2 was not observed in all of the DDFT samples examined in our study; in humans high levels of variability in immunostaining have been reported between different
416
F. Beccati et al./The Veterinary Journal 205 (2015) 413–416
specimens and between different sections of individual specimen (Danielson et al., 2007; Bjur et al., 2008). Finally, the results of TH and α1-AR immunostaining of tenocytes in zone 2 were consistent. This highlights evidence of catecholamine production and a functional catecholamine response system in tenocytes; it also suggests that sympathetic innervation in the tendon might contribute to a catecholaminergic autocrine/paracrine system that might be relevant to the development of degenerative conditions. Furthermore, it was of interest to note the presence of α 1 -AR in scattered spindle cells in zone 1, consistent with fibroblast-like cells. The presence of fibroblasts, which have been shown to express catecholamine receptors, indicates a connecting role in the development of the autocrine/paracrine system (Ackermann, 2013). The role of sympathetic innervation in the physiology of the equine DDFT in the digit remains unclear. In humans, studies have suggested that adrenergic stimulation of tendons might be involved in the proliferation of tenocytes, endothelial cells, and nerve cells (Ackermann, 2013). Furthermore, the adrenergic stimulation of fibroblast-like cells could contribute to cell proliferation and/or apoptosis (Ackermann, 2013). Sympathetic activity might be modified in horses with distal DDFT tendinopathy and further studies of sympathetic activity in the equine DDFT are warranted to determine whether TH or α1-AR may be altered in samples with tendinopathy.
Conflict of interest statement None of the authors of this review has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
Acknowledgements An abstract of this paper was presented as a Short Communication (Hippozorg Award) at the European Veterinary Conference 2014, Amsterdam, 17–19 April 2014. The authors would like to acknowledge the precious technical assistance of Mrs. Gabriella Mancini and Dr. Emilia Del Rossi, and Mrs Lucia Antinori for her support in the collection of samples. References Ackermann, P.W., 2013. Neuronal regulation of tendon homoeostasis. International Journal of Experimental Pathology 94, 271–286. Bjur, D., Danielson, P., Alfredson, H., Forsgren, S., 2008. Immunohistochemical and in situ hybridization observations favor a local catecholamine production in the human Achilles tendon. Histology and Histopathology 23, 197–208. Blunden, A., Dyson, S., Murray, R., Schramme, M., 2006. Histopathology in horses with chronic palmar foot pain and age-matched controls. Part 2: The deep digital flexor tendon. Equine Veterinary Journal 38, 23–27. Bolen, G., Busoni, V., Jacqmot, O., Snaps, F., 2007. Sonographic anatomy of the palmarodistal aspect of the equine digit. Veterinary Radiology & Ultrasound 48, 270–275. Bordeaux, J., Welsh, A., Agarwal, S., Killiam, E., Baquero, M., Hanna, J., Anagnostou, V., Rimm, D., 2010. Antibody validation. Biotechniques 48, 197–209. Butler, J.A., Colles, C.M., Dyson, S.J., Kold, S.E., Poulos, P.W., 2008. Foot, pastern and fetlock. In: Clinical Radiology of the Horse, Third Ed. Wiley-Blackwell, Oxford. UK, pp. 102–128. Danielson, P., Alfredson, H., Forsgren, S., 2007. Studies on the importance of sympathetic innervation, adrenergic receptors, and a possible local catecholamine production in the development of patellar tendinopathy (tendinosis) in Man. Microscopy Research and Technique 70, 310–324. Russo, D., Bombardi, C., Grandis, A., Furness, J.B., Spadari, A., Bernardini, C., Chiocchetti, R., 2010. Sympathetic innervation of the ileocecal junction in horses. The Journal of Comparative Neurology 19, 4046–4066. Schramme, M.C., 2011. Deep digital flexor tendonopathy in the foot. Equine Veterinary Education 23, 403–415. Scott, A., Bahr, R., 2009. Neuropeptides in tendinopathy. Frontiers in Bioscience 14, 2203–2211.
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Short Communication
Sympathetic innervation of the suprasesamoidean region of the deep digital flexor tendon in the forelimbs of horses F. Beccati *, M. Pepe, L. Pascucci, P. Ceccarelli, E. Chiaradia, F. Mancini, M.T. Mandara Centro di Studi del Cavallo Sportivo, Dipartimento di Medicina Veterinaria, Università degli Studi di Perugia, Via San Costanzo 4, 06126 Perugia, Italy
A R T I C L E
I N F O
Article history: Accepted 4 June 2015 Keywords: Horse Deep digital flexor tendon Tyrosine hydroxylase Alpha-1 adrenergic receptor Sympathetic innervation
A B S T R A C T
The purpose of this study was to delineate the pattern of sympathetic innervation in the suprasesamoidean region of the deep digital flexor tendon (DDFT) in horses using immunohistochemical staining (IHC) for tyrosine hydroxylase (TH) and alpha-1 adrenergic receptor (α1-AR). Fourteen forelimbs were collected from 10 horses. Longitudinal sections of the suprasesamoidean region of healthy DDFTs were harvested. Most of the sympathetic innervation was found to be in the walls of blood vessels. The tendon tissue proper was sparsely innervated, with a lesser degree of innervation within the dorsal fibrocartilage. Increased α1-AR immunostaining was also detected in walls of blood vessels and in spindle cells of fibrocartilage. Both α1-AR and TH immunostaining were detected in tenocytes. These findings support the presence of autocrine/paracrine catecholaminergic signalling in equine tendon tissue. © 2015 Elsevier Ltd. All rights reserved.
Progress in the field of diagnostic imaging has shown that the deep digital flexor tendon (DDFT) (M. flexor digitalis profundus) is one of the most commonly injured structures in sport horses undergoing magnetic resonance imaging for foot pain, with a total prevalence ranging from 30% to 83% (Schramme, 2011). The macroscopic appearance of the tendon as well as its histological structure and various pathohistological lesions has been previously described (Blunden et al., 2006). Blunden et al. (2006) suggested that DDFT lesions have a degenerative rather than inflammatory pathogenesis. The peripheral nervous system participates in the regulation of cell proliferation, release of cytokines and growth factors, modulation of inflammation and immune responses, and hormone release (Ackermann, 2013). It has, therefore, been proposed that abnormalities in neurotransmitter function might be related to the degenerative events in tendinosis in humans (Scott and Bahr, 2009). The nerves innervating tendons are mostly composed of nonmyelinated, slow-transmitting Aγ-, Aδ-, B-, and C-fibres with a small fraction of myelinated, fast-transmitting Aα- and Aβ-fibres. Mechanoreception, nociception, and vasomotor modulation are three primary functions of tendon innervation (Ackermann, 2013). Autonomic innervation is involved in the dynamic regulation of tendon blood flow and includes parasympathetic and sympathetic components that mediate vasodilatation and vasocostriction, respectively (Scott and Bahr, 2009). Tyrosine hydroxylate (TH) is the rate-limiting enzyme in sympathetic neuropeptide synthesis and alpha-adrenergic
* Corresponding author. Tel.: +39 075 585 7713. E-mail address: [email protected] (F. Beccati). http://dx.doi.org/10.1016/j.tvjl.2015.06.004 1090-0233/© 2015 Elsevier Ltd. All rights reserved.
receptors (α1-AR) are the main autonomic receptors expressed in tendon (Scott and Bahr, 2009) thus representing the main biomarkers for sympathetic innervation in tendons. Although tendons have traditionally been considered as hypoinnervated, data regarding the innervation pattern of the DDFT in horses are scant. The aim of this study was to determine the pattern of sympathetic innervation in the suprasesamoidean region of the normal equine DDFT in the forelimb using immunohistochemistry (IHC) and TH and the α1-AR as markers of different functional phases of the same autonomic system. Fourteen feet were collected from 10 horses with no history of forelimb-foot pain that had been humanely euthanased for reasons not related to this study. Signalment details of horses are presented in Table 1. Post-mortem, radiographic and ultrasonographic examinations of the feet were performed to verify that the DDFT was of normal appearance and as previously described (Bolen et al., 2007; Butler et al., 2008). To exclude the presence of tendon lesions, gross and histological examinations of the DDFT were also performed. All procedures were approved by the Institutional Animal Care Committee of the University of Perugia. Tissue samples were collected immediately after death and the suprasesamoidean region of the DDFT was excised. For each DDFT, three full-thickness samples were selected for the histological and IHC examinations (Fig. 1). Samples were fixed in 10% neutralbuffered formalin, dehydrated and embedded in paraffin wax; 4 μm thick sections were stained with Harris’s haematoxylin and eosin, Masson’s trichrome stain, aldehyde fuchsin, and Verhoeff’s stain to characterise tissue morphology. Immunohistochemical staining was performed using the 1B5 mouse anti-human tyrosine hydroxylase monoclonal antibody (TH; 1:40; Leica-Novocastra) and a rabbit antihuman α1-AR polyclonal antibody (1:80; antibodies-online).
414
F. Beccati et al./The Veterinary Journal 205 (2015) 413–416
Table 1 Summary of the signalment details of the animals used in this study.
1 2 3 4 5 6 7 8 9 10
Breed
Age
Sex
Previous use
Warmblood Warmblood Anglo-Arab Anglo-Arab Arab Warmblood Anglo-Arab Pony Thoroughbred Thoroughbred
14 21 15 22 16 13 23 21 7 3
Female Gender Female Female Gender Gender Gender Gender Female Female
Show jumping Show jumping Show jumping General purpose Endurance Show jumping Endurance General purpose General purpose Race
tendon, consisting of dense connective tissue with fibres oriented in a longitudinal fashion and rich in blood vessels. The anti-human α1-AR antibodies were validated for detection of the equine α1-AR protein in the IHC by Western blotting (WB) according to Bordeaux et al. (2010). Briefly, protein samples, obtained from the equine liver (20 μg), brain (20 μg), and tendon (40 μg), were separated by 12% polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred to nitrocellulose membranes, probed with anti-human α1-AR antibody (1:500) and detected with horseradish peroxidase conjugated goat anti-rabbit IgG polyclonal antibody (Santa Cruz Biotechnology). The immunoreactive bands were visualised with ECL reagents and by exposing X-ray films. The WB analysis (Fig. 2) indicated that the anti-human α1-AR antibody used is specific for the detection of equine α1-AR protein because a single band corresponding to approximately the expected molecular weight of the α1-AR (~50 kDa) protein was detected in the liver, brain and tendon line. Further, no extraneous bands were detected (Bordeaux et al., 2010). Based on previous studies in equine nervous tissue, WB validation for TH was not performed (Russo et al., 2010). Zone 1 was scantly vascularised, except for the superficial subsynovial stratum. The distal region of this layer contained collagen fibres that ran at angles to one another and numerous isolated or grouped chondrocytes (Fig. 3a). No TH immunoreactivity was observed (Fig. 3d) while a remarkable cytoplasmic immunoreactivity for α1-AR was detected in scattered spindle cells (Fig. 3g). Zone 2 was characterised by an extensive inter-fascicular vascular network that extended throughout the loose connective tissue of the endotendon (Fig. 3b). Tenocytes were the most common type of cells and were arranged in long parallel rows among collagen fibres. Slight to remarkable TH-immunoreactivity was detected, consisting of occasional positive nerve fascicules and tenocytes (not
Fig. 1. Diagram to show the sites (black dotted lines) for collection of samples of the suprasesamoidean region (white dotted lines) of the deep digital flexor tendon for histological and immunohistochemical examinations. SNC, synovium of the navicular bursa.
After deparaffinisation and rehydration, the endogenous peroxidase was inactivated by incubating the sections in 3% hydrogen peroxide (for TH) or methanol (for α1-AR) at room temperature. The slides were probed with the primary antibodies overnight at 4 °C. Antigen retrieval was not performed. Immunoreactivity was detected using an avidin–biotin method (LSAB+/System-HRP, DakoCytomation) and aminoethyl carbazole as the substrate (AEC + Substrate-Chromogen Ready-to-use, DakoCytomation). Carazzi’s haematoxylin was used as a counterstain. Paramount mounting medium (DakoCytomation) was used to mount the coverslips on the slides. For each tendon sample, immunolabelling staining intensity was assessed as one of three grades (absent, slight and remarkable). The equine midbrain was used as a positive control in the IHC experiments, as indicated by the manufacturers. Negative controls were carried out in the same manner with omission of the primary antibody. For the histomorphological and IHC analyses, the DDFT sections were divided into three different zones according to Blunden et al. (2006): (1) zone 1, the most dorsal layer of the DDFT (approximately 30% of the entire tendon thickness) consisting of dense irregular connective tissue and rich in elastic fibres; (2) zone 2, primarily dense regular connective tissue, exhibiting closely packed longitudinal collagen bundles; (3) zone 3, the palmar layer of the
Fig. 2. Validation of α1-AR antibody for equine proteins. Total protein extract from equine liver (20 μg), brain (20 μg) and tendon (40 μg) were analysed by Western blotting to test the specificity of the antibody. A single band corresponding to approximately the expected molecular weight of the α1-AR (~50 kDa) protein was detected in each line. No extraneous bands were detected.
F. Beccati et al./The Veterinary Journal 205 (2015) 413–416
415
a
b
c
d
e
f
g
h
i
Fig. 3. Equine deep digital flexor tendon (DDFT). Normal morphology (a–c) and sympathetic system (d–i). (a) Zone 1. Fibrocartilaginous tissue with well differentiated nest of chondrocytes (Masson’s trichrome stain, ×20); (b) Zone 2. Dense regular connective tissue rich in tenocytes and endotendon fibrovascular septa (H&E, ×20); (c) Zone 3. Blood vessels of different diameter embedded in abundant connective tissue (Masson’s trichrome stain, ×20); (d) Zone 1. No elements of this zone are positive for TH (ABCmethod and Carazzi’s haematoxylin, ×20); (e) Zone 2. Marked TH-immunoreaction showed by small blood vessel of endotendon septa (ABC-method and Carazzi’s haematoxylin, ×20); (f) Zone 3. The TH-immunoreaction markedly occurs in media–adventitia layers of blood vessels (ABC-method and Carazzi’s haematoxylin, ×20); (g) Zone 1. Scattered spindle cells show a marked cytoplasmic α1-AR immunoreaction (ABC-method and Carazzi’s haematoxylin, ×20); (h) Zone 2. Marked α1-AR immunoreaction expressed by tenocytes (ABC-method and Carazzi’s haematoxylin, ×20); (i) Zone 3. Cellular elements of vascular media–adventitia layers express a marked α1-AR immunoreaction (ABCmethod and Carazzi’s haematoxylin, ×20).
shown); in two cases, a remarkable level of TH-immunoreactivity was associated with the blood vessels of the endotendon septa (Fig. 3e). Foci of remarkable immunoreactivity for α1-AR were sparsely distributed in the walls of the blood vessels in the endotendon septa and in the tenocytes (Fig. 3h). In the tendon proper tissue, positive nerve fibres showed remarkable immunoreactivity for α1-AR. Zone 3 was rich in blood vessels (Fig. 3c). The IHC staining for TH ranged from slight to remarkable. The TH immunoreactivity pattern indicated the presence of nerve profile in the proximity of blood vessels, and in a large extent located at media–adventitia borders (Fig. 3f). The IHC staining for α1-AR was slight to remarkable in the smooth muscle cells of the tunica media of the arterioles (Fig. 3i). The results imply a number of interpretations. Firstly, TH immunoreactivity associated with the blood vessels, particularly as observed in zone 3, is consistent with previous studies of TH in the patellar tendon (Danielson et al., 2007) and the Achilles tendon (Bjur et al., 2008) in humans. Secondly, the sympathetic innervation
detected in the blood vessels and nerve fascicules in zone 2 was less extensive than that observed in zone 3, although THimmunoreactivity was occasionally observed in the tenocytes; recent studies in humans have shown that tendon neurotransmitters, including catecholamines, may be produced by tenocytes (Scott and Bahr, 2009). Thirdly, the complete absence of TH-immunoreactivity in zone 1 is consistent with findings observed in previous human studies (Bjur et al., 2008), which showed that the level of innervation decreased with increasing distance from the loose connective tissue. Fourthly, the pattern of sympathetic innervation determined in IHC analysis for the TH was confirmed in the IHC analysis of α1-AR immunoreactivity in the tunica media of arterioles scattered throughout zone 3 and in the walls of blood vessels in zone 2, further indicating the presence of sympathetic nerve fibres. However, the α1-AR immunoreactivity in zone 2 was not observed in all of the DDFT samples examined in our study; in humans high levels of variability in immunostaining have been reported between different
416
F. Beccati et al./The Veterinary Journal 205 (2015) 413–416
specimens and between different sections of individual specimen (Danielson et al., 2007; Bjur et al., 2008). Finally, the results of TH and α1-AR immunostaining of tenocytes in zone 2 were consistent. This highlights evidence of catecholamine production and a functional catecholamine response system in tenocytes; it also suggests that sympathetic innervation in the tendon might contribute to a catecholaminergic autocrine/paracrine system that might be relevant to the development of degenerative conditions. Furthermore, it was of interest to note the presence of α 1 -AR in scattered spindle cells in zone 1, consistent with fibroblast-like cells. The presence of fibroblasts, which have been shown to express catecholamine receptors, indicates a connecting role in the development of the autocrine/paracrine system (Ackermann, 2013). The role of sympathetic innervation in the physiology of the equine DDFT in the digit remains unclear. In humans, studies have suggested that adrenergic stimulation of tendons might be involved in the proliferation of tenocytes, endothelial cells, and nerve cells (Ackermann, 2013). Furthermore, the adrenergic stimulation of fibroblast-like cells could contribute to cell proliferation and/or apoptosis (Ackermann, 2013). Sympathetic activity might be modified in horses with distal DDFT tendinopathy and further studies of sympathetic activity in the equine DDFT are warranted to determine whether TH or α1-AR may be altered in samples with tendinopathy.
Conflict of interest statement None of the authors of this review has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
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