Journal of Clinical Neuroscience (2003) 10(2), 219–223 0967-5868/03/$ - see front matter ª 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0967-5868(02)00336-3 Laboratory studies The effects of calcium channel antagonist nimodipine on end-plate vascularity of the degenerated intervertebral disc in ratsq,qq Mehmet Turgut1 MD, Aysegül Uysal2 PHD, Serap Uslu2 MSC, Namık Tavus3 1 MD, Mine Ertem Yurtseven2 PHD 2 Department of Neurosurgery, Adnan Menderes University Medical Faculty, 09100 Aydın, Turkey, Department of Histology and Embryology, Ege University Medical Faculty, 35100 _Izmir, Turkey, 3Ege BT Diagnostic Centre, 09020 Aydın, Turkey Summary The vascular channels at the end-plate of the intervertebral disc are very important in maintaining a healthy disc. With age, a reduction of the nutrition of the avascular nucleus pulposus is inevitable. On the other hand the calcium channel antagonist nimodipine has been shown to have a positive effect on blood flow in the region of the vertebral end-plate. To evaluate the effects of nimodipine on the endplate vascularity in the degenerative discs, we have produced an experimental disc degeneration and evaluated the radiological and histopathological features of the end-plate of the degenerated discs. Adult rats were divided into 3 groups: control ðn ¼ 5Þ, operated degeneration ðn ¼ 5Þ, and nimodipine treatment (n ¼ 5). Using a posterior approach, a cut was made parallel to the end-plates in the posterior annulus fibrosus in 5 consecutive intervertebral discs between the 5th and 10th vertebral segments of the tails of adult Swiss Albino rats. At 8 weeks, 5 of these animals were treated with nimodipine. In each experimental group, 1 animal was examined using computed tomography (CT) to study the density of the cartilage end-plate of the disc. Then, the animals were sacrificed for subsequent histopathological evaluation. We found that the vascular channel counts and percentage areas from animals treated with nimodipine were higher than from both the non-operative control and operated degeneration groups, although these were not statistically different. Accordingly, the profile of the density histogram in the nimodipine-treated group showed a wide plateau, indicating an increase in the vascularity in this region. From our results, we suggest that nimodipine enhances vascularisation of the cartilage end-plate in the disc. It is possible that the increased proportion of vascular contacts at the end-plate has a beneficial effect in the nutrition of the disc. However, further experimental studies will be needed to determine the validity of this statement in animals or human beings. ª 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cartilage end-plate, intervertebral disc, nimodipine, vascular channels INTRODUCTION Both in animals and humans, the intervertebral disc consists of the nucleus pulposus, the annulus fibrosus, and the end-plate.1 The end-plates that lie at the cranial and caudal interface of the disc are the major structural component of the disc.1;2 Anatomically, it is important to consider that the end-plate consists of a cartilage component and an osseous component.1 Vascular channels in the end-plate of the intervertebral disc are particularly vital for maintaining the nutrition of the avascular nucleus pulposus.2–4 There are reports that these channels disappear with disc degeneration and eventually become obliterated by calcification.1;5;6 On the other hand, calcium channel antagonists have been shown to have a variety of potential effects in improving blood flow.7–10 These include enhancement of blood flow, selective dilatation of vessels, inhibition of vascular contraction, and stimulation of new vessel growth.11 There is no doubt that re-establishment of vascular supply to the intervertebral disc may constitute a new avenue. The purpose of the present study is to reproduce experimental disc degeneration for studying the radiological and histopathological features, and to evaluate the effects of calcium channel antagonist nimodipine on end-plate vascularity of the degenerated intervertebral disc. In other words, will the nimodipine administration to rats exert an action to the vessel channels of the cartilage end-plate region such as new vessel growth? MATERIALS AND METHODS q This study was presented in part at the 37th National Annual Neurological Congress, Antalya, 31 October–4 November, 2001. qq Author contributions: The authors indicated in the title made substantial contributions to the following tasks of research: initial conception (M.T.); design (M.T., A.U., N.T.); administrative, technical, or material support (M.T., N.T., M.E.Y.); acquisition of data (M.T., A.U., S.U., N.T.); laboratory analysis and interpretation of data (M.T., A.U., S.U., N.T.); drafting of the manuscript (M.T., S.U.); critical revision of the manuscript for important intellectual content (M.T., A.U., N.T., M.E.Y.). The views expressed herein are those of the authors and not necessarily their institions or sources of support. Received 25 March 2002 Accepted 24 June 2002 Correspondance to: Dr. Mehmet Turgut, Cumhuriyet Mahallesi, Cumhuriyet Caddesi, No: 2/D Daire:7, TR-09020 Aydın, Turkey. Tel.: +90-256-2134874; Fax: +90-256-2132172; E-mail: drmturgut@yahoo.com Experimental protocol A total of 15 adult Swiss Albino rats (each weighing between 120 and 160 g) of either sex were used in this experiment. All experiments were performed according to the guidelines for the ethical treatment of animals of the European Union, and animal protocols were approved by the Laboratory Animal Care Committee of Ege University Hospital. The animals were anaesthetised with a combination of 10 mg/kg xylasine (BAYER Birlesßik Alman I_ lacß Fabrikaları T.A.Sß ., I_ stanbul) and 60 mg/kg ketamine hydrochloride (Parke-Davis, I_ stanbul) given intramuscularly. Additional doses were given when needed. The rats were divided randomly into 3 groups (5 animals in each) according to the experimental procedures. The first non-operated control group consisted of 5 sham animals. 219 220 Turgut et al. Fig. 1 Photograph taken following posterior surgical approach. Note that the wires are incl uded to identify the 5 consecutive disc interspaces operated in rat’s tail. Surgical procedure In the other 2 groups (operated degeneration and nimodipine treatment), under aseptic conditions the spine of the ratÕs tail was exposed using a posterior approach, and in 5 consecutive intervertebral discs between the 5th and 10th vertebral segments of the tails, a cut was made parallel to the end-plates in the posterior annulus fibrosus (Fig. 1). Following surgery, all wounds were closed in a standard manner with absorbable sutures. At 8 weeks, 5 of these animals (nimodipine treatment group) were treated with daily intubations of calcium-blocking pharmaceutical nimodipine (15 mg/kg) for 4 weeks. Since nimodipine is insoluble, it was administered orally by intragastric intubation as suspension. The drug was suspended in 2% aqueous methyl cellulose solution and administered by intragastric injection through plastic neonatal feeding tubes. Computed tomography scanning In each experimental group, 1 animal was randomly selected for computed tomography (CT) examination using a Hitachi W450 CT scanner to study the density of the cartilage end-plate of the intervertebral disc. The profile of the density histograms is related to the sum of the pixels that have the same scale of gray in the CT image. The white pixels represent the osseous tissue, the gray pixels the cartilaginous tissue, and the black pixels water or air. If white (or gray or black) pixels are prevalent, the profile of the histogram will be spike-shaped. If white, gray, and black pixels are balanced, the profile will be plateau-shaped.12 For this reason, the histogram concerning the normal cartilage end-plate has a spike profile, which indicates a prevalently homogenous cartilaginous tissue. In contrast, the plateau profile is typical of an endplate in which vascular areas and calcified tissue are quantitatively balanced. The animals were sacrificed for subsequent histopathological evaluation. Histopathological studies All animals were studied histopathologically. In each animal, the spine including 5 consecutive intervertebral discs between the 5th and 10th vertebral segments of the tails was fixed in 10% neutral buffered formalin and was then decalcified in 5% hydrochloric acid. A single midsagittal section was done in each specimen and each slice was processed into paraffin wax using standard methods. Tissue sections of 5 lm thickness were stained Journal of Clinical Neuroscience (2003) 10(2), 219–223 Fig. 2 Density histograms of animals in non-operated control (A), operated degeneration (B), and nimodipine treatment (C) groups. Note ‘‘plateau profile’’ in nimodipine-treated group, indicating increased vascularisation of the cartilage end-plate of the degenerated intervertebral disc. with haematoxylin and eosin for histological examination and the vascularity of the cranial and caudal cartilage end-plate regions was evaluated separately by histologists unaware of the experimental group. The vascular channels in both cartilage endplates of each animal in all groups were counted. For each intervertebral disc, the area of vascular channel was quantified as the percentage of the total cartilage end-plate area as described elsewhere.13 Data analysis Mean values and standard deviations of mean were determined. One-way analysis of variance (ANOVA) test was used to deterª 2003 Elsevier Science Ltd. All rights reserved. Nimodipine on end-plate vascularity of disc in rats 221 Table 1 Vascular channel counts in the cartilage end-plate regions of the intervertebral discs No. of animals VCCs  SD  Non-operated control group Operated degeneration group Nimodipine treatment group 5 11:17  2:49 5 20:98  3:19 5 22:96  3:42 Abbreviations: VCC, vascular channel count; SD, mean standard deviation. Table 2 Percentage area of vascular channels in the cartilage end-plate regions of the intervertebral discs No. of animals Area of VCs  SD ð%Þ  Non-operated control group Operated degeneration group Nimodipine treatment group 5 33:29  9:40 5 39:75  8:37 5 40:16  10:88 Abbreviations: VC, vascular channel; SD, mean standard deviation. generation group, the histogram showed a spike profile (Figs. 2A and B). On the other hand, the profile of the density histogram in nimodipine-treated group showed a wide plateau, indicating an increase in the amount of lower density tissues in this region (Fig. 2C). Histopathological findings Fig. 3 The histological appearance of cartilage end-plate regions of nonoperated control HE, original magnification 126 (A); operated degeneration HE, original magnification 126 (B); and nimodipine treated HE, original magnification 126 (C) rats. Note that the value of vascular channel counts was highest in animals treated with nimodipine. mine the significance of any differences between the groups. It was followed by DuncanÕs post-hoc test for pairwise comparisons. Statistical significance was P ¼ 0:05. RESULTS Radiological findings In this study, CT densitometric analysis was used to demonstrate increased vascularisation in the end-plates of the intervertebral disc. In non-operated control group as well as the operated deª 2003 Elsevier Science Ltd. All rights reserved. Vascular channel counts. Morphologically, the vessel counts of the cranial and caudal end-plates of all groups were measured. In operated degeneration group, the vessel counts were significantly higher than from the control values of the non-operated animals ðP < 0:05Þ (Figs. 3A and B). In nimodipine-treated animals, the counts were found to be increased from a mean value of 20:98  3:19 in operated degeneration group to a mean value of 22:96  3:42 (Fig. 3C). However, the difference in values between the animals in the 2 groups was not statistically significant (P > 0:05; Table 1). Area of vascular channels. The total percentage area of vascular channels to cartilage end-plate of all groups were calculated (Table 2). The average value of percentage area of vascular channels was higher in nimodipine-treated animals than both nonoperated control and operated degeneration groups, suggesting that nimodipine stimulated the vascularisation of cartilage endplate of the disc, but there was no statistically significant difference between the values of animals in operated degeneration and nimodipine treatment groups ðP > 0:05Þ. DISCUSSION During the early stage of the growth, the blood vessels within the vertebral end-plates provide nutrition of the intervertebral disc and then the discs become avascular as a result of aging.1;5;6 In the adult the intervertebral discs are reliant on exchange of nutrients through the end-plates in maintaining the mechanical function. As a rule, the central end-plate is permeable, while the lateral regions of the end-plate are impermeable.4;14 Thus, the vascular channels at the end-plate appear to be a critical consideration in maintaining a healthy disc. Degenerative diseases of the spine constitute a public health problem in the world thereby they need for better understanding of the pathogenesis and exploring new avenues for therapy. So far, various experimental models have been described to produce disc Journal of Clinical Neuroscience (2003) 10(2), 219–223 222 Turgut et al. degeneration.15–23 Some authors have used bipedal rats to reproduce the human situation.23 Others have used direct action on the intervertebral disc by traumatic damage to the annulus fibrosus.15;19 In every model the disc height is slightly reduced, as in human disc herniations. This diminution in height is associated with protrusions and the typical intervertebral disc herniation is posterior due to variation in structure of the fibers, or associated with altered biomechanics, or different types of insertion of the annular fibers into the peripheral vertebral border.3;19;20;23–27 Typically, vascular channels at the end-plate proliferate to maintain adequate nutrition of the disc.1;28 It has been claimed that the inducing of new blood vessels in the end-plate is facilitated by the activation of enzymes such as matrix metalloproteinases.29;30 As indicated earlier, with advancing age the cartilage component of the end-plate has undergone mineralisation and the vascular channels within the end-plate become occluded by calcification, preventing the diffusion of solutes into the disc.1 In other words, the cartilage component of the end-plate becomes an osseous tissue with age in association with an increase of the density of the end-plate. Recently, type X collagen is considered to be a potential cause of these observations.31 It is evident that re-establishment of blood flow in the region of the end-plate is necessary in degenerative discs. On the other hand, it has been stated that calcium antagonists have a variety of potential effects in improving blood flow and ischemic disturbances.7–10 The present investigation was undertaken to study the effects of calcium channel antagonist nimodipine on end-plate vascularity of the degenerated intervertebral disc. Although our study is limited by the histopathological and radiological studies, to authorsÕ knowledge, no study on the effects of nimodipine for vascularisation of endplate of the intervertebral disc exists. In our study, the vessel counts of the cartilage end-plate were found to be higher in animals treated with nimodipine when compared with animals in both non-operated control and operated degeneration groups. This has important clinical significance, although the difference between the values of nimodipine-treated group and operated degeneration group was not significant statistically. Indeed, the therapeutic window and dosage of nimodipine also needs to be tested in future studies. We think that the value of the vascular channels area is more reliable than the value of the vessel count for studying the effects of calcium channel antagonist nimodipine on end-plate vascularity of the degenerated intervertebral disc in rats. This model is valid in the study of the effects of nimodipine on endplate vascularity of the degenerated intervertebral disc. It seems likely that the rat is a useful experimental model for the investigation of the effects of nimodipine on degenerated intervertebral disc and that the calcium channel blocker has a beneficial effect on the end-plate vascularity of the intervertebral disc. Our data on density histograms of the animals in all groups were compatible with the histopathological findings. Profile of the density histogram of the cartilage end-plate in nimodipine-treated group showed a wide plateau, indicating an increase in the amount of vascular channels with lower density in this region, in contrast to a spike profile in other groups. Although the potential mechanism of such an effect is unclear, it appears that nimodipine enhances vascularisation of the cartilage end-plate in degenerated intervertebral disc. Thus, the increased proportion of vascular contacts at the end-plate will provide the means for the transport of osteoblast precursors, producing osteoblast activation in the vicinity of the end-plate region. We would therefore expect vascularisation of cartilage end-plate and osteoblasts to result in increased mechanical strength. Journal of Clinical Neuroscience (2003) 10(2), 219–223 In conclusion, the end-plate is important in disc nutrition and the mechanical function of the spine. Nimodipine administration to rats has a positive effect on vascularisation of the end-plate in the degenerated disc. This study requires confirmation by other researchers, but opens up a new area for experimental and clinical investigation of the vascular channels at the vertebral end-plate. Future research will involve the use of microangiography and immunohistochemical study to assess the role of nimodipine on the vascularity of end-plate in degenerative discs. It is possible that such an evaluation may play a crucial role in explaining the reduced bone formation in the vertebra and diminished vascular channels in various destructive procedures such as laser disectomy and radiofrequency application to the disc tissue. ACKNOWLEDGEMENTS The study medication was partially donated by BAYER AG (6077). The authors thank Mr. I_ smail Zonguldak, Mr. B€ ulent Ata, € zdemir, and Mr. Feyzi Subasßı for their technical Mrs. Fatma O € st€ un for the statistical assistance and comments and Mrs. Hatice U analysis. REFERENCES 1. Moore RJ. The vertebral end-plate: what do we know?. Eur Spine J 2000; 9: 92–96. 2. Chandraraj S, Briggs CA, Opeskin K. Disc herniations in the young and endplate vascularity. Clin Anat 1998; 11: 171–176. 3. Holm S, Maroudas A, Urban JPG et al. Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res 1981; 8: 101–119. 4. Nachemson A, Lewin T, Maroudas A et al. In vitro diffusion of dye through the endplates and the annulus fibrosus of human intervertebral discs. Acta Orthop Scand 1970; 41: 589–607. 5. Roberts S, Menage J, Urban JPG. Biochemical and structural properties of the cartilage endplate and its relation to the intervertebral disc. Spine 1989; 14: 166–174. 6. Twomey LT, Taylor JR. Age changes in lumbar vertebrae and intervertebral discs. Clin Orthop 1987; 224: 97–104. 7. Faden AI, Jacobs TP, Smith MT. Evaluation of the calcium channel antagonist nimodipine in experimental spinal cord ischemia. J Neurosurg 1984; 60: 796– 799. 8. Guha A, Tator CH, Piper I. Increase in rat spinal cord blood flow with the calcium channel blocker, nimodipine. J Neurosurg 1985; 63: 250–259. 9. Mabe H, Takagi T, Umemura S et al. Effect of calcium entry blocker, nimodipine, on the cerebral function and metabolic recovery following experimental cerebral ischemia. Brain Nerve 1985; 37: 1067–1072. 10. Scriabine A, Battye R, Hoffmeister F. Nimodipine. In: Scriabine A (ed) New Drugs Annual: Cardiovascular Drugs. Raven Press, New York 1985; 197–218. 11. Haws CW, Heistad DD. Effects of nimodipine on cerebral vessels: role of extracellular calcium in vasoconstrictive responses. Stroke 1983; 14: 122 (abstract). 12. Zagra A, Lamartina C, Pace A et al. Computer-assisted tomography of scoliosis operated with or without HarringtonÕs rod. Biomechanics aspects of the fusion. Spine 1990; 15: 796–802. 13. Kalichman MW, Powell HC, Myers RR. Quantitative histologic analysis of local anesthetic-induced injury to rat sciatic nerve. J Pharmacol Exp Ther 1989; 250: 406–413. 14. Maroudas A, Stockwell RA, Nachemson A et al. Factors involved in the nutrition of the human lumbar intervertebral disc: cellularity and diffusion of glucose in vitro. J Anat 1975; 120: 113–130. 15. Brinckmann P, Porter RW. A laboratory model of lumbar disc protrusion. Spine 1994; 19: 228–235. 16. Lane-Petter W, Pearson AEG. In: The Laboratory Animal: Principles and Practice. Academic Press, London 1971; 43–62. 17. Latorre A, Albareda J, Castiella T et al. Experimental model of multidirectional disc hernia in rats. Int Orthop 1998; 22: 44–48. 18. Lindblom K. Experimental ruptures of intervertebral disc in ratÕs tails. J Bone Joint Surg [Am] 1952; 34: 123–128. 19. Lipson SJ, Muir H. Experimental intervertebral disc degeneration. Morphologic and proteoglycan changes over time. Arthritis Rheum 1981; 24: 12–21. ª 2003 Elsevier Science Ltd. All rights reserved. Differences in responses to nociceptive 20. Neufeld JH, Machado T, Margelin L. Variables affecting disc size in the lumbar spine of rabbits: anesthesia, paralysis and disc injury. J Orthop Res 1991; 9: 104–112. 21. Stokes IA, Counts DF, Frymoyer JW. Experimental instability in rabbit lumbar spine. Spine 1989; 14: 68–72. 22. Takenaka Y, Revel M, Kahan A et al. Experimental model of disc herniations in rats for study of nucleolytic drugs. Spine 1987; 12: 556–560. 23. Yamada K. The dynamics of experimental posture. Experimental study of intervertebral disc herniation in bipedal animals. Clin Orthop 1962; 25: 20–31. 24. Adams MA, Hutton WC. Prolapsed intervertebral disc. A hyperflexion injury. Spine 1982; 7: 184–191. 25. Trueta J. The role of vessels in osteogenesis. 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Journal of Clinical Neuroscience (2003) 10(2), 223–225 0967-5868/03/$ - see front matter ª 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0967-5868(02)00332-6 Differences in responses to nociceptive stimulation of the oral and aboral oesophagus Thomas Hummel1 MD, Sibylle Barz2 MD, € lscher2,3 Tobias Ho MD, Winfried L Neuhuber4 MD 1 Department of Otorhinolaryngology, University of Dresden Medical School, Fetscherstr. 74, 01307 Dresden, Germany, 2Department of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-N€urnberg, Krankenhausstr. 9, 91054 Erlangen, Germany, 3Department of Nuclear Therapy, University of Dresden Medical School, Fetscherstr. 74, 01307 €rnberg, Krankenhausstr. 9, Dresden, Germany, 4Department of Anatomy I, University of Erlangen-Nu 91054 Erlangen, Germany Summary The present study examined the role of vagal innervation of the rat esophagus in nociception. Electromyographic recordings from neck muscles were used as indicators of pseudoaffective reactions in lightly anaesthetized animals; responses were obtained during mechanical (distension) and chemical (HCl) nociceptive stimulation of the upper cervical (4.5 cm from incisors) and midthoracic (7 cm from incisors) esophagus. Compared to midthoracic esophageal stimulation, stimulation of the upper esophagus produced more vigorous responses. Bilateral transections of the cervical vagus and superior laryngeal nerves led to a gradual diminution of responses to upper stimulation, while the same manoeuvre increased responses to lower esophagus stimulation. These results suggest a contribution of vagal afferents to pseudoaffective responses elicited by noxious stimulation of the upper esophagus. ª 2003 Elsevier Science Ltd. All rights reserved. Keywords: pain, nociception, vagus nerve, visceral INTRODUCTION MATERIALS AND METHODS Visceral nociception and pain is classically considered to be mediated by spinal afferent pathways.1;2 This assumption is based on clinical evidence and on the fact that intensity coding and high threshold visceral afferents are found in ‘‘sympathetic’’ nerves projecting to the thoracolumbar spinal cord. Conversely, ‘‘parasympathetic’’, in particular vagal afferents are thought to serve largely non-nociceptive reflex regulation of inner organs. However, recent experiments have demonstrated high threshold vagal afferents from the rat stomach;3 in addition, upon visceral noxious stimulation much higher c-Fos expression was found in central termination areas of ‘‘parasympathetic’’ compared to ‘‘sympathetic’’ afferents.4–6 To assess the relative contribution of vagal and spinal afferents to nociception in the esophagus, we used the alternative approach of recording pseudoaffective responses to noxious visceral stimulation (compare7 ) combined with nerve transections. Surgical procedure Received 13 June 2002 Accepted 31 August 2002 Correspondence to: Thomas Hummel, MD, Department of Otorhinolaryngology, University of Dresden Medical School, Fetscherstr. 74, 01307 Dresden, Germany. Tel.: +49-351-458-4189; Fax: +49-351-458-4326; E-mail: thummel@rcs.urz.tu-dresden.de ª 2003 Elsevier Science Ltd. All rights reserved. Experiments were performed in eight male Sprague–Dawley rats (Charles River, Germany) weighing between 450 and 600 g. Experiments were approved by the Animal Care and Use Committee of the local government of Middle Franconia/Bavaria. Light anaesthesia was induced by pentobarbital sodium (NembutalÓ, 45– 50 mg/kg i.p., Sanofi, Germany) and maintained throughout the experiment with a constant intravenous infusion (12 mg/kg/h). The right femoral vein and artery were canulated for drug administration and the monitoring of arterial blood pressure, respectively (Biometrics, Germany). The electromyogram (EMG) was recorded from neck muscles using needle electrodes (E2 platinum alloy electrodes, Grass, West Warwick, RI, USA) and continuously monitored, both visually (HAMEG oszilloscope, type 410, Germany) and acoustically (auditory monitor, Grass, West Warwick, RI, USA). Mean arterial blood pressure and EKG were continuously monitored (cardiovascular monitor, Science Products, Germany). The trachea was cannulated for artificial ventilation. Body temperature was maintained by a hot water heating pad (36 °C) and overhead lamps. Animals breathed spontaneously during the experiments. The left and right cervical vagus were carefully dissected from surrounding tissue, and both superior laryngeal nerves were exposed. Journal of Clinical Neuroscience (2003) 10(2), 223–225