Incidence and diagnostic relevance of urinary peptides and proteins in dogs with renal disease and urolithiasis

Titelblatt, Inhaltsverzeichnis, Abkürzungen, Lebenslauf, Danksagung, Eidesstattliche Erklärung 1\. Einleitung 2\. Literaturübersicht 3\. Material und Methoden 4.1. Ergebnisse Niereninsuffiziente Hunde 4.2.Ergebnisse Harnsteinpatienten 5\. Diskussion Zusammenfassung Summary Tabellenanhang LiteraturverzeichnisAufgrund der Beobachtung, dass Störungen der Nierenfunktion häufig mit einer Veränderung der renalen Proteinausscheidung einhergehen, hat die Analyse von Harnproteinen in den letzten Jahren besonders in der klinischen Diagnostik an Bedeutung gewonnen. Veränderungen im tubulointerstitiellen Bereich der Niere werden mitunter für das Fortschreiten von Nierenversagen verantwortlich gemacht, unabhängig von der zugrunde liegenden Ursache. Die tubuläre Proteinurie bzw. Enzymurie, welches die Zeichen für eine tubuläre Schädigung sind, werden beim Menschen bei verschiedenen Formen von Nierenerkrankungen beobachtet. Der Western-Blot und andere immunologische Methoden sind die mit Erfolg beim Menschen eingesetzten Techniken, um Harnproteine zu identifizieren. Die Identifikation von Proteinen kann das Verständnis der physiologischen und pathophysiologischen Abläufe in der Niere vorantreiben und damit zur Entdeckung von neuen Markern bei verschiedenen Erkrankungen führen. Trotz intensiver Forschung ist der Kenntnisstand zur Pathogenese der Harnsteinbildung beim Hund, welche die Basis für jede sinnvolle Steinbehandlung darstellt, eher dürftig. Der normale Harn ist generell vor Nukleation, Wachstum und Aggregation geschützt. Der Harn von Steinbildnern ist dagegen reich an Promotern, bzw. arm an Inhibitoren. Dies erklärt die Tendenz gegenüber den Gesunden, eine höhere Anzahl an Kristallen auszuscheiden, die sich zu größeren Aggregaten zusammenklumpen können. Makromoleküle des Harns spielen bei der Entstehung von Harnsteinen eine wichtige modifizierende Rolle, d.h. die Proteine scheinen den größten Anteil der inhibitorischen Kraft zu besitzen. Gegenstand der klinisch-analytischen prospektiven Studie war die Untersuchung des Harnproteinprofils sowie Veränderungen im Vitamin-A-Stoffwechsel bei zwei verschiedenen Erkrankungen des caninen Harnapparates: die renal bedingte Niereninsuffizienz und die Urolithiasis. Dazu wurden Blut und Harn von 19 Hunden mit Niereninsuffizienz, von 25 Hunden mit Harnsteinen und 21 Kontrollhunden untersucht. Außerdem wurden die Nierengewebeproben von 4 nierenkranken Hunden und die Harnsteine der 25 Steinpatienten analysiert. Die Vitamin-A-Bestimmung (Retinol und Retinylester) erfolgte mittels Umkehrphasen-Hochdruck-Flüssigkeits-Chromatographie (RP- HPLC). Die Vitamin-A-Trägerproteine Retinol-Bindungsprotein (RBP) und Tamm- Horsfall Protein (THP) wurden quantitativ im Blut und Harn durch einen ELISA bestimmt und ihr qualitativer Nachweis erfolgte nach elektrophoretischer Trennung im Western-Blot. Der Harn wurde zusätzlich auf das Vorkommen weiterer Harnproteine wie Immunglobulin G, Transferrin, Albumin und Vitamin-D-Bindungsprotein (DBP) und deren diagnostische Bedeutung für die Nierenfunktion untersucht. Die Ergebnisse wurden mit den dazugehörigen Harnmustern nach ihrer Auftrennung mittels SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE) verglichen. Zusätzlich wurden die Gewebeschnitte der Nieren auf RBP, THP und Megalin immunhistologisch untersucht. Um den Effekt von THP und Vitamin A auf die Harnsteinbildung zu überprüfen, wurde der Harn von 10 gesunden Hunden mit unterschiedlich konzentrierten Calciumchloridlösungen behandelt. Nach Entfernen des Präzipitates wurden die Konzentrationen von THP, Retinol und Retinylester im Überstand gemessen. Die organische Matrix wurde nach Extraktion der 25 Harnsteine sowohl mit der herkömmlichen Methode der SDS-PAGE als auch mit einer neuen Technik, der sogenannten "Surface enhanced laser desorption/ionization - time-of-flight" Massenspektrometrie (SELDI-TOF MS) analysiert. Zudem wurde der Harn aller Hunde auf ihr unterschiedliches Harnproteinprofil mit Hilfe der SELDI-TOF MS untersucht. Vitamin A konnte im Blutserum und im Harn aller untersuchten Hunde in Form von Retinol und Retinylestern nachgewiesen werden. Patienten mit Niereninsuffizienz (NI) zeigten signifikant höhere Serumkonzentrationen an Retinol sowie an den Retinylestern Oleat und Palmitat. Zudem fielen die NI Hunde durch eine gesteigerte Retinolausscheidung im Harn auf, dokumentiert durch eine höhere fraktionelle Clearance von Retinol (2,96 gegenüber 0,15). Im Gegensatz zu gesunden Hunden schieden die erkrankten Tiere RBP im Harn aus. Dagegen war keine Veränderung der Serumwerte von RBP zwischen beiden Gruppen festzustellen. Bei dem Vergleich mit den Kontrolltieren zeigten die nierenkranken Hunde eine signifikant reduzierte Ausscheidung von THP über den Harn. Das Muster der immunologisch nachgewiesenen Harnproteine korrelierte sehr gut mit den Harnmustern, die durch die elektrophoretische Auftrennung erhalten wurden. Darüber hinaus konnten mit Hilfe der Immunhistologie Defekte in der Rezeptorexpression des proximalen Tubulus, dem Megalin, dargestellt werden sowie ein pathologisches Muster beim Nachweis von RBP und THP. Die Ergebnisse korrelierten mit den Befunden der Histologie. Durch die Anwendung der SELDI-TOF Massenspektrometrie war es möglich, zusätzliche Peptide und Proteine zu identifizieren, die für das Vorhandensein einer Niereninsuffizienz typisch sein können. Die Hunde mit Harnsteinen unterschieden sich nicht signifikant in ihrem Vitamin-A-Metabolismus von der Kontrollgruppe. Einzig eine leichte Erhöhung des Serumretinols war feststellbar, während im Harn die Vitamin-A-Konzentrationen wie bei den Gesunden eine weite Spanne aufwiesen. In den meisten Fällen konnte RBP in Spuren im Harn der Urolithiasispatienten nachgewiesen werden. Deutlich waren die geringeren Harnkonzentrationen von THP bei Hunden mit Harnsteinen. Unter dem Einwirken der Calciumchloridlösungen schien THP sein Potential als Inhibitor der Harnsteinbildung durch die Bindung zu den Retinylestern einzuschränken. Die Harnanalyse von Gesunden gegenüber den Steinpatienten zeigte durch die Anwendung der SELDI-TOF MS signifikante Unterschiede, obwohl insgesamt eine weite Heterogenität der Harnprofile festzustellen war. Im Vergleich zu gesunden Tieren war die Menge spezifischer Harnproteine bei Steinbildnern unterschiedlich. Typische elektrophoretische Proteinmuster von verschiedenen Harnsteinextrakten zeigten Proteinbanden bei 31 und 14 kDa. Ein Protein bei 66 kDa (Albumin) erschien als Hauptbestandteil in allen Steinarten. Weitere Peptide und Proteine der organische Matrix konnten mittels SELDI-TOF MS identifiziert werden. Die Ergebnisse der Studie dokumentieren einen Einfluss der Nierenerkrankungen auf den Stoffwechsel von Retinol in Blut und Harn von Hunden. Normalübliche Teststicks zur Erkennung einer Proteinurie oder auch die Bestimmung des Protein/Kreatinin - Verhältnisses im Harn sind nicht ausreichend für eine genaue Information der verbleibenden Nierenfunktion in Hinblick auf Ultrafiltration bzw. Rückresorption. Mit Hilfe der Untersuchungen konnte gezeigt werden, dass die Anwendung spezifischer in der Humanmedizin bekannter Markerproteine insbesondere RBP und THP zur Diagnostik einer Nierenfunktionsstörung auch für den Hund sinnvoll ist. Die Bestimmung von RBP bietet einen sehr sensitiven Marker für Schädigungen des proximalen Tubulusabschnitts in der Niere. Eine reduzierte Ausscheidung von THP weist dagegen auf eine Störung des distalen Abschnitts hin. Ferner konnte gezeigt werden, dass eine neue Methode der SELDI-TOF MS den Vorteil bietet, einer Vielzahl an Harnproteinen gleichzeitig zu untersuchen und damit neue potentielle Marker aufzudecken. Die Untersuchung des Vitamin-A-Stoffwechsels lässt keinen Zusammenhang mit der Bildung von Harnsteinen erkennen. Bei den untersuchten Hunden konnte aber eine signifikante Abnahme der renalen THP-Sezernierung bei Steinbildnern gemessen werden. Die Ergebnisse lassen vermuten, dass THP, wie beim Menschen, eine Bedeutung als Regulator bei der Aggregation von Kristallen hat. Möglicherweise ist für die Steinbildung von größerer Bedeutung, dass die Ausscheidung von THP als Antwort auf eine Zunahme der Calcium- und Oxalatkonzentration nur bei gesunden Menschen ansteigt. Der protektive Mechanismus von THP scheint bei Steinbildnern zu fehlen, wie in anderen Studien berichtet. Neben THP konnten weitere Proteine mit Hilfe der SELDI-TOF Massenspektrometrie ermittelt werden, die eine mögliche Bedeutung für die Lithogenese zeigen. Die Möglichkeit, die Expression vieler Harnproteine gleichzeitig zu messen, bietet einen signifikanten technischen Vorteil. Die Methode der SELDI-TOF MS scheint für die Suche nach Markern bei der Urolithiasis geeignet. Zum ersten Mal wurde die Zusammensetzung der organischen Matrix von Hundeharnsteinen untersucht.Defects of renal function is often followed by a change of urinary protein excretion. In the last years the investigation of urinary proteins has become more and more of diagnostic relevance. It is proposed that tubulointerstitial changes play an important role in the progression of renal failure irrespective of the underlying etiology. Tubular proteinuria and enzymuria, markers of tubular dysfunction, have been reported in humans with different forms of renal diseases. Western blotting and other immunological methods are techniques employed to identify urinary proteins. Identification of urinary proteins may lead to an enhanced understanding of renal physiology and pathopyhsiology, and thus lead to the discovery of novel markers for diseases. Despite intense research the knowledge of stone pathogenesis, which is the basis of every rational stone metaphylaxis, has remained rather scanty. Urine is normally protected from nucleation, growth and aggregation. Urine of stone formers, however, is supposedly rich in promoters, or deficient in inhibitors, thus explaining its tendency to excrete crystals in greater quantities and clustered into larger aggregates than those in the urine of healthy subjects. Urinary macromolecules play an important modifying role in stone formation and proteins appear to be responsible for most of this inhibitory activity. The objective of this clinical analytical prospective study was to investigate the changes in the metabolism of vitamin A and their carrier proteins in plasma and urine in two different diseases - canine renal insufficiency and urolithiasis. Blood and urine were collected from 19 dogs with renal disease, 25 dogs with urolithiasis and 21 healthy dogs. In addition, renal tissue samples were obtained from 4 dogs with renal disease. Uroliths from the 25 dogs were analysed. Retinol and retinyl esters were determined using a reversed-phase high performance liquid chromatography system (rp-HPLC). The carrier proteins retinol binding protein (RBP) and Tamm-Horsfall protein (THP) were measured quantitatively in blood and urine by an enzyme-linked immunosorbent assay (ELISA) and for qualitative determination the detection was performed after electrophoretical separation by Western blotting. Besides RBP and THP, the urinary excretion of further urinary proteins like immunglobulin G, transferrin, albumin and vitamin D binding protein (DBP) were assessed by Western blotting for their diagnostic relevance of renal function. The results were compared with the urinary protein patterns from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the tissue sections of the kidneys were investigated for RBP, THP and Megalin by immunohistology. In case of urolithiasis the urine of ten healthy dogs were treated with different solutions of calcium chloride to determine a possible role of THP and vitamin A in lithogenesis. Therefore, the concentrations of THP, retinol and retinyl esters were analyzed after decantation. To investigate the protein composition of the organic matrix of the 25 urinary stones the proteins extracted from the stones were submitted to SDS-PAGE and also to a novel approach, the surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Finally, an analysis of the urinary protein patterns of all dog groups was performed with SELDI technique. Vitamin A was present as retinol and retinyl esters in blood plasma as well as in urine of all dogs. In patients with renal disease (RD) serum concentrations of retinol, retinyl oleate and palmitate were significantly higher than the values found in healthy dogs. An enhanced urinary excretion of retinol became evident in RD dogs documented as a higher fractional clearance of retinol with 2,96 vs. 0,15. In contrast to healthy dogs, RBP was detected in the urine of the RD patients. Serum levels of RBP, however, did not change significantly with renal disease. When compared with controls, a significant reduction of the renal THP secretion was found for RD dogs. The immunological detection of the other urinary proteins showed the same pattern of excretion with the electrophoretic separation on SDS-PAGE. Furthermore, immunohistochemical investigations of the kidneys showed defects of the proximal receptor megalin expression and of renal handling with RBP and THP. They correlated well with the pathohistological findings. With SELDI-TOF mass spectrometry it was possible to identify further peptides and proteins which were characteristic for canine renal insufficiency. Dogs with urolithiasis (U) did not differ significantly in their vitamin A metabolism when compared with controls. Nevertheless, serum retinol was modestly elevated in U dogs while urinary vitamin A levels varied widely, as it was observed in the control urine. In most instances urinary RBP was detected but only in trace amounts, whereas serum RBP did not change with urolithiasis. The group of U dogs differed significantly from controls in a lower urinary THP. Moreover, the inhibitory activity of THP for stone formation was reduced with the binding to retinyl esters under the different calcium chloride solutions. With SELDI-TOF mass spectrometry a reflection on urine between stone patients and healthy dogs represented significant differences although the urinary protein profiles consisted of wide heterogeneity. Stone formers excreted different amounts of certain proteins when compared with healthy dogs. In typical electrophoretic patterns of proteins extracted from urinary stone matrix of the four types of stones a protein at 31 and 14 kDa appeared. A 66 kDa protein appeared to be by far as the major constituent on all types of stones. The SELDI-TOF mass spectrometry offered further peptides and proteins for each stone type. Results of this study has shown that renal disease affects the concentrations of retinol in plasma and urine of dogs. Commercial dipstick urinalysis for proteinuria or urine protein to creatinine values have been used to screen renal disease, but these methods do not provide accurate information to determine the onset to functional renal impairment. The results of this study in dogs with different renal diseases and a wide range of renal function indicate that especially RBP and THP may be appropriate markers for use in the diagnosis of tubular proteinuria in dogs. Measurements of urinary RBP provides a highly sensitive indicator of proximal renal tubular malfunction. The reduction of urinary THP pointed to the dysfunction of the distal tubules. Further, SELDI-TOF MS can be used successfully in rapid screening of a number of urinary proteins indicated as putative markers for renal dysfunction. Analyzing the vitamin A metabolism in canine urolithiasis shows that no influence on lithogenesis becomes apparent. The results of a decreased urinary THP excretion in dogs with uroliths indicate that THP plays a role as a modifier of crystal aggregation. Possibly most relevant to stone formation is the fact that THP excretion rises only in healthy subjects in response to increasing urinary calcium and oxalate concentrations, whereas this self- protective mechanism appears to be missing in stone formers as it is reported in other studies. Beside THP other urinary proteins may play an important role in lithogenesis of dogs. The possibility of examining the expression of the majority of urinary proteins simultaneously represents a significant technical advance. SELDI-TOF mass spectrometry seems to be a useful method for biomarker discovery in urolithiasis. In this study, for the first time the protein composition of the organic matrix of canine uroliths were investigated

Characterisation of transthyretin and retinol-binding protein in plasma and cerebrospinal fluid of dogs

The aim of this study was to investigate differences in concentrations of vitamin A, transthyretin (TTR) and retinol-binding protein (RBP) between plasma and cerebrospinal fluid (CSF) in dogs. RBP was detected using ELISA, and both RBP and TTR by Western blot analysis after separation on SDS-PAGE. Vitamin A was determined by high performance liquid chromatography. RBP and TTR as well as vitamin A were detected in all samples but at substantially lower concentrations in CSF compared to plasma. RBP in dog plasma showed a similar molecular mass to that of humans, whereas canine TTR had a lower molecular mass. Comparison between plasma and CSF showed that both RBP and TTR were of lower molecular mass in CSF. In CSF, RBP and retinol were present at 10-100-fold lower concentrations compared to plasma. Retinyl esters were present only in minute amounts in 5/17 samples. In conclusion, the CSF of dogs compared to humans is significantly different in terms of both quality and quantity of transport proteins for vitamin A

Investigation of Sciatic Nerve Surgical Anatomy in Dogs and Cats: A Comparative Cadaveric Study

Objective: Dogs and cats with traumatic or iatrogenic partial sciatic nerve lesions frequently have disparate clinical signs. Cats commonly walk with a plantigrade posture in the affected pelvic limb, which is rarely observed in dogs. We hypothesized that the tibial nerve would be localized more laterally in cats and medially in dogs, and that the tibial nerve would be larger than the peroneal nerve in cats, which may result in a greater susceptibility of the tibial nerve to iatrogenic trauma in cats. Goal of the present cadaveric study was to investigate differences present in pelvic sciatic nerve anatomy between dogs and cats. Methods: This is an anatomic cadaveric study. Dogs (n=7) and cats (n=7); n=28 hindlimbs. A simple suture was placed without nerve mobilization on the lateral aspect of the lumbosacral trunk at the level of the mid-body of the ilium. A caudolateral approach to the femur was then performed. The lumbosacral trunk was transected in the intrapelvic area cranial to the suture marking the lateral aspect. The peroneal and tibial branches of the sciatic nerve were separated. The proximal lateral knot was identified as being part of the tibial or peroneal nerve, respectively, and the diameter of the tibial and peroneal branches at the level of the suture (mid-ilium) were measured. Results: No difference in relative size of the tibial compared to the peroneal nerve was found between dogs and cats. The tibial nerve was not found to be localized lateral to the peroneal nerve more frequently in cats compared to dogs. Conclusions: Findings suggest that the intrapelvic anatomy of the lumbosacral trunk cannot fully explain the plantigrade posture observed in cats with traumatic or iatrogenic partial sciatic nerve injury

In vitro effect of different mediators of apoptosis on canine cranial and caudal cruciate ligament fibroblasts and its reversibility by pancaspase inhibitor zVAD.fmk

Primary fibroblast cultures of canine cranial (CCL) and caudal (CaCL) cruciate ligaments were stimulated with different apoptosis inducers with or without preincubation of the pancaspase inhibitor zVAD.fmk. In contrast to CaCL fibroblasts, fibroblasts from CCL were significantly more susceptible to apoptosis inducers of the intrinsic pathway like doxorubicin, cisplatin and nitric oxide (NO)-donors and to Fas ligand (FasL), an apoptosis inducer of the death receptor pathway. Apoptotic response to staurosporine and the peroxynitrite donor GEA was similar in both ligament fibroblasts. Stimulation with dexamethasone or TNFalpha could not induce apoptosis in CCL and CaCL fibroblasts, in spite of present TNFR1 and TNFR2 receptors. zVAD.fmk was able to prevent apoptosis in up to 66% of CCL cells when treated with FasL, cisplatin or doxorubicin but it had no effect on NO or peroxynitrite induced apoptosis. In conclusion, differential susceptibility to apoptotic triggers like FasL or NO between cranial and caudal cruciate ligament fibroblasts in vitro may be a reflection of the different susceptibilities to degenerative rupture of the ligament. These findings indicate that a general caspase inhibition does not completely protect canine CCL cells from apoptosis

Nitric oxide induces cell death in canine cruciate ligament cells by activation of tyrosine kinase and reactive oxygen species

Abstract Background There is increasing evidence suggesting that development of progressive canine cranial cruciate ligament (CCL) rupture involves a gradual degeneration of the CCL itself, initiated by a combination of factors, ranging from mechanical to biochemical. To date, knowledge is lacking to what extent cruciate disease results from abnormal biomechanics on a normal ligament or contrary how far preliminary alterations of the ligament due to biochemical factors provoke abnormal biomechanics. This study is focused on nitric oxide (NO), one of the potential biochemical factors. The NO-donor sodium nitroprusside (SNP) has been used to study NO-dependent cell death in canine cranial and caudal cruciate ligament cells and to characterize signaling mechanisms during NO-stimulation. Results Sodium nitroprusside increased apoptotic cell death dose- and time-dependently in cruciate ligamentocytes. Cells from the CCL were more susceptible to apoptosis than CaCL cells. Caspase-3 processing in response to SNP was not detected. Testing major upstream and signal transducing pathways, NO-induced cruciate ligament cell death seemed to be mediated on different levels. Specific inhibition of tyrosine kinase significantly decreased SNP-induced cell death. Mitogen activated protein kinase ERK1 and 2 are activated upon NO and provide anti-apoptotic signals whereas p38 kinase and protein kinase C are not involved. Moreover, data showed that the inhibition reactive oxygen species (ROS) significantly reduced the level of cruciate ligament cell death. Conclusions Our data support the hypothesis that canine cruciate ligamentocytes, independently from their origin (CCL or CaCL) follow crucial signaling pathways involved in NO-induced cell death. However, the difference on susceptibility upon NO-mediated apoptosis seems to be dependent on other pathways than on these tested in the present study. In both, CCL and CaCL, the activation of the tyrosine kinase and the generation of ROS reveal important signaling pathways. In perspective, new efforts to prevent the development and progression of cruciate disease may include strategies aimed at reducing ROS.</p

Nitric oxide induces cell death in canine cruciate ligament cells by activation of tyrosine kinase and reactive oxygen species

BACKGROUND: There is increasing evidence suggesting that development of progressive canine cranial cruciate ligament (CCL) rupture involves a gradual degeneration of the CCL itself, initiated by a combination of factors, ranging from mechanical to biochemical. To date, knowledge is lacking to what extent cruciate disease results from abnormal biomechanics on a normal ligament or contrary how far preliminary alterations of the ligament due to biochemical factors provoke abnormal biomechanics. This study is focused on nitric oxide (NO), one of the potential biochemical factors. The NO-donor sodium nitroprusside (SNP) has been used to study NO-dependent cell death in canine cranial and caudal cruciate ligament cells and to characterize signaling mechanisms during NO-stimulation. RESULTS: Sodium nitroprusside increased apoptotic cell death dose- and time-dependently in cruciate ligamentocytes. Cells from the CCL were more susceptible to apoptosis than CaCL cells. Caspase-3 processing in response to SNP was not detected. Testing major upstream and signal transducing pathways, NO-induced cruciate ligament cell death seemed to be mediated on different levels. Specific inhibition of tyrosine kinase significantly decreased SNP-induced cell death. Mitogen activated protein kinase ERK1 and 2 are activated upon NO and provide anti-apoptotic signals whereas p38 kinase and protein kinase C are not involved. Moreover, data showed that the inhibition reactive oxygen species (ROS) significantly reduced the level of cruciate ligament cell death. CONCLUSIONS: Our data support the hypothesis that canine cruciate ligamentocytes, independently from their origin (CCL or CaCL) follow crucial signaling pathways involved in NO-induced cell death. However, the difference on susceptibility upon NO-mediated apoptosis seems to be dependent on other pathways than on these tested in the present study. In both, CCL and CaCL, the activation of the tyrosine kinase and the generation of ROS reveal important signaling pathways. In perspective, new efforts to prevent the development and progression of cruciate disease may include strategies aimed at reducing ROS

Evaluation of apoptotic cell death in normal and chondrodystrophic canine intervertebral discs

Disc degeneration occurs commonly in dogs. A variety of factors is thought to contribute an inappropriate disc matrix that isolate cells in the disc and lead to apoptosis. Disc herniation with radiculopathy and discogenic pain are the results of the degenerative process. The objective of this prospective study was to determine the extent of apoptosis in intact and herniated intervertebral discs of chondrodystrophic dogs and non-chondrodystrophic dogs. In addition, the nucleus pulposus (NP) was histologically compared between non-chondrodystrophic and chondrodystrophic dogs. Thoracolumbar intervertebral discs and parts of the extruded nucleus pulposus were harvested from 45 dogs. Samples were subsequently stained with haematoxylin-eosin and processed to detect cleaved caspase-3 and poly(ADP-ribose) polymerase. A significant greater degree of apoptosis was observed in herniated NPs of chondrodystrophic dogs compared to non- chondrodystrophic dogs with poly (ADP-ribose) polymerase and cleaved caspase- 3 detection. Within the group of chondrodystrophic dogs, dogs with an intact disc and younger than 6 years showed a significant lower incidence of apoptosis in the NP compared to the herniated NP of chondrodystrophic dogs. The extent of apoptosis in the annulus fibrosus was not different between the intact disc from chondrodystrophic and non- chondrodystrophic dogs. An age-related increase of apoptotic cells in NP and annulus fibrosus was found in the intact non-herniated intervertebral discs. Histologically, absence of notochordal cells and occurrence of chondroid metaplasia were observed in the nucleus pulposus of chondrodystrophic dogs. As a result, we found that apoptosis plays a role in disc degeneration in chondrodystrophic dogs