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Radiological Investigation of Guinea Pig (Cavia porcellus) Lumbar Vertebral Morphology ‒ A Biomechanical Aspect

Radiological Investigation of Guinea Pig (Cavia porcellus) Lumbar Vertebral Morphology ‒ A... Acta Veterinaria-Beograd 2023, 73 (1), 55-70 UDK: 599.324.7:591.471.35 DOI: 10.2478/acve-2023-0005 Research article RADIOLOGICAL INVESTIGATION OF GUINEA PIG (CAVIA PORCELLUS) LUMBAR VERTEBRAL MORPHOLOGY ‒ A BIOMECHANICAL ASPECT 1 1 2 Marko Jumake MITROVIĆ *, Sara KITANOVIĆ , Nikola TATALOVIĆ , 1 1 Anastasija TODOROVIĆ , Mirjana LAZAREVIĆ MACANOVIĆ University of Belgrade, Faculty of Veterinary Medicine, Department of Radiology and Radiation Hygiene, Bulevar oslobođenja 18, Belgrade, Serbia; University of Belgrade, Institute for Biological Research “Siniša Stanković” ‒ National Institute of Republic of Serbia, Department of Physiology, Bulevar Despota Stefana, Belgrade, Serbia (Received 17 September, Accepted 22 December 2022) Numerous studies are based on the use of animal models; however, in bipedal and tetrapedal organisms there are signifi cant differences in the biomechanics of the spinal column, which can signifi cantly impair the quality and applicability of the results obtained. The aim of this study is to obtain basic data on the morphometric parameters of guinea pig lumbar vertebrae, the analysis of which will indicate the location of the biggest mechanical load. The lumbar vertebra morphometry test was performed by means of X-ray imageing obtained from 12 guinea pigs, with equal numbers of males and females. The results of investigations show that guinea pig lumbar vertebrae have an irregular trapezoid geometry and that the measured body lengths of L4 and L5 are the largest. The height parameters determined in the medial level showed that L4 had the most concave body. Moreover, L4 had the greatest depth of the spinal canal at the same measurement level. Consequently, in guinea pigs, the greatest load is in the L4 region, unlike in humans, where, due to the axial load of the spinal column, the highest pressure is exerted on the last lumbar vertebrae. Keywords: biomechanics, guinea pig, lumbar spine, morphometry, radiology INTRODUCTION Knowledge of biomechanics enables a better understanding of the role of bones and associated soft tissues in stabilizing the spine, as well as an understanding of the destabilizing effects that can occur due to the appearance of tumors, traumatic and degenerative damages [1]. In a functional sense, bone and soft tissue structures are closely connected and their mutual action achieves the stability of the spine and its mobility in all three axes [2]. *Corresponding author: e-mail: markom@vet.bg.ac.rs 55 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 In humans, who are bipedal organisms, the thoracic part of the spine has limited mobility because ribs are attached to the vertebrae at one end and to the sternum at the other end. Unlike the thoracic segment, the mobility of the lumbar spine is signifi cantly higher and at the same time, it bears the greatest axial load [2]. It is known that bones adapt to the action of mechanical forces by changing their morphology [3], so the size of the thoracic vertebrae in humans increases from the fi rst to the last [1], and this trend continues distally so that the last lumbar vertebra has the greatest length, height and width [4]. Considering the high incidence of various pathological conditions in the spinal column of humans, which predominantly affect the lumbar region, numerous studies are aimed at fi nding an adequate biomechanical model to examine the static-mechanical relationships of lumbar vertebrae. In the available literature, there are various fi ndings of different morphometric and/or biomechanics examinations of the spinal column performed on rabbits [5], dogs [6], pigs [7], sheep [8] and cattle [9,10]; however, the search for an optimal animal model is still ongoing. It should be borne in mind that there are signifi cant differences in the biomechanics of the spinal columns of bipedal and tetrapedal organisms (Supplementary Figure S1), which can signifi cantly impair the quality and applicability of the results obtained. Supplementary Figure S1: Presentation of the morphometry of the spinal column in different species of mammals. Guinea pigs (Cavia porcellus) are a herbivorous species of rodents from the family Caviidae which due to their low body weight, short reproductive cycle, calm temperament, as well as anatomical and physiological characteristics, are increasingly used as models in various studies [11]. In neurology and orthopedics, guinea pigs have been used to examine ischemic lesions in the spinal cord [12], and also for the study of spontaneous and induced osteoarthritis [13]. The spinal column of rodents has a total of 26 presacral vertebrae, composed of 7 cervical and 19 thoracolumbar vertebrae [14]. The cervical spine region of mammals is characterized by the smallest variations 56 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect in the number of vertebrae, which is usually attributed to the pleiotropic function of Hox genes. In addition, variations in the number of cervical vertebrae are associated with an increased risk of prenatal mortality and neonatal cancer [14,15]. On the other side, the thoracolumbar region of the spine of guinea pigs shows greater variability, and in most individuals, it is composed of 13 thoracic and 6 lumbar vertebrae [16]. The aim of this study was to obtain data on the morphometric parameters of the lumbar vertebrae of guinea pigs and to view them from the aspect of biomechanics in order to obtain information on the mechanical load on the lumbar spine of these animals. MATERIAL AND METHODS Animals The examination was performed on 12 guinea pigs (Cavia porcellus), with an equal number of males (6) and females (6), body weight 380-800 g. All morphometric examinations of the lumbar vertebrae were performed on X-rays (permission no. 323-07-0850112019-05 issued by the Veterinary Administration of the Ministry of Agriculture and Environmental Protection of the Republic of Serbia). Experimental procedures were performed in compliance with Directive 2010/63/EU on the protection of animals used for experimental and other scientifi c purposes. Preparation for radiological examination In order to minimize the guinea pigs’ movements and to obtain images of optimal quality for analysis, all animals were anesthetized following a three-hour deprivation of food and water. Animals were anesthetized using ketamine hydrochloride (Ketamidor 10%, Richter Pharma, Austria) in a dose of 60 mg/kg body weight, with xylazine hydrochloride premedication (Xylased, Bioveta, Czech Republic) in a dose of 4 mg/ kg. The application was performed subcutaneously, using a 25 G-diameter injection needle. Radiological examination All radiographs were performed using a ZooMax Gold X-ray machine (Control-X Medical, Hungary). The animals were placed in a plexiglass basket, and morphometric examinations of various parameters of the lumbar vertebrae were performed only on images made in lateral recumbency in the laterolateral projection (Figure 1A) because in the ventrodorsal projection it was not possible to accurately measure the width of the vertebral body due to summation of shadows of gastrointestinal contents and bony outlines of vertebrae. The X-rays were performed at a focus fi lm distance of 90 cm, using a voltage of 44 kVp, while the exposure was 5 mAs. 57 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Morphometric examinations of lumbar vertebrae Morphometric examinations of all six lumbar vertebrae (L1-6) of guinea pigs were performed in the RadiAnt DICOM Viewer program by three examiners. The length of vertebral bodies was measured at three different levels: dorsal (L ), medial (L ) and D M ventral (L ), as well as the height: cranial (H ), medial (H ) and caudal (H ). The V CR M CA depth of the spinal canal was measured in the central part of each examined vertebra (Figure 1B-D). Figure 1. Lateral recumbency of guinea pig in a plexiglass basket (A) and morphometric examinations of lumbar vertebrae: (B) measuring the length in the dorsal (LD), medial (LM) and ventral (LV) parts of the vertebral body; (C) measuring height in the cranial (HCR), medial (HM) and caudal (HCA) parts of the body; (D) measuring the depth of the spinal canal (DSC). Data analysis and statistical procedures Morphometric parameters of lumbar vertebrae (length and height of vertebral bodies at three different levels and depth of the spinal canal) are presented as mean ± standard deviation (SD) in millimeters. All individual values are presented in the Supplementary Material. The sample size (n) was 12. Differences in the length and height of vertebrae bodies in all three levels were tested by factorial analysis of variance (two-way ANOVA) with the number of vertebrae and the level of measurement as factors, and post hoc compared by Tukey’s honest signifi cant difference (HSD) test. Differences in depth of the spinal canal were tested by single factor analysis of variance (one-way ANOVA) 58 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect and post hoc compared by Tukey’s HSD test. Correlations between body weight and the morphometric parameters of lumbar vertebrae were examined using Pearson’s correlation coeffi cient as well as linear regression of morphometric parameters as the dependent variable on body weight as the independent variable. Deviations in linear regression curve slopes from zero were tested by the F-test. Comparisons of linear regression curve slopes were also performed using the F-test. Statistical signifi cance of the correlation coeffi cient was tested using Student’s t-test. The signifi cance level was 0.05. All analyses were conducted using GraphPad Prism 8 (GraphPad Software Inc.). RESULTS Length of lumbar vertebral bodies Two-way ANOVA showed a signifi cant difference in the lengths of different lumbar vertebrae (F=15.4; p<0.0001) as well as a signifi cant difference between the different levels of measurement, namely dorsal (L ), medial (L ) and ventral (L ) (F=8.754; D M V p<0.001) (Figure 2A, Supplementary Tables S 1 and S2). Supplementary table S1: Length of lumbar vertebral bodies (in millimeters); L – length in the dorsal level; L – length in the medial level; L ‒ length in the ventral level. M V L1 6.567 6.870 6.903 7.380 7.320 7.133 8.260 9.177 8.070 8.527 9.133 8.320 L2 7.160 7.390 7.460 8.170 8.000 7.843 8.980 10.167 8.823 9.383 9.870 8.963 L3 7.557 7.927 7.963 8.737 8.420 8.417 9.573 10.733 9.400 10.100 10.567 9.557 L4 8.047 8.263 8.293 9.217 8.893 8.893 10.000 11.233 9.960 10.633 11.000 9.990 L5 7.797 8.073 8.220 9.303 8.670 9.403 9.700 11.233 10.100 11.133 11.100 9.777 L6 6.787 7.067 7.073 8.230 7.767 8.840 8.387 10.097 8.863 10.667 10.153 8.480 L1 6.317 6.417 6.450 6.857 7.320 6.690 7.743 8.830 7.560 8.167 8.847 7.657 L2 6.820 6.977 7.137 7.777 7.820 7.507 8.517 9.643 8.473 9.023 9.600 8.540 L3 7.437 7.627 7.617 8.253 8.097 8.173 9.120 10.233 9.020 9.710 10.247 9.023 L4 7.780 7.993 8.103 8.897 8.793 8.573 9.620 10.833 9.577 10.267 10.733 9.507 L5 7.690 7.863 8.003 9.170 8.590 9.117 9.497 10.967 9.803 10.933 10.900 9.423 L6 6.797 7.000 7.020 8.263 7.720 8.630 8.407 10.100 8.670 10.233 9.947 8.273 L1 5.597 6.003 5.943 6.537 6.457 6.247 7.587 8.560 7.097 7.913 8.573 7.337 L2 6.357 6.483 6.693 7.477 7.090 6.650 8.350 9.210 7.900 8.473 9.143 8.017 L3 7.170 7.440 7.263 8.067 7.563 7.427 8.883 9.743 8.830 8.903 9.897 8.833 L4 7.393 7.810 7.797 8.457 8.010 8.003 9.173 10.367 9.400 10.067 10.600 9.267 L5 7.373 7.397 7.390 8.590 7.917 8.690 8.783 10.300 9.180 10.467 10.600 9.133 L6 6.060 6.507 6.610 7.450 7.087 8.087 7.883 9.600 7.890 9.757 9.587 7.973 59 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Figure 2. (A) Length of guinea pig lumbar vertebrae bodies in dorsal, medial and ventral levels. (B) Linear regression of lumbar vertebrae bodies lengths as the dependent variable on body weight as the independent variable. LD ‒ length in the dorsal part of the vertebral body; LM ‒ length in the medial part of the vertebral body; LV ‒ length in the ventral part of the vertebral body; p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; n.s. – nonsignifi cant. 60 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect Supplementary table S2: Height of lumbar vertebral bodies (in millimeters); H – height CR in the level of cranial epiphysis; H – height in the medial level; H – height in the level of M CA caudal epiphysis. CR L1 3.003 2.950 2.977 2.573 2.833 3.473 3.483 3.100 3.643 2.973 3.023 3.583 L2 2.887 3.003 2.945 2.750 2.899 3.327 3.307 3.400 3.697 3.173 2.930 3.500 L3 2.910 3.257 3.083 2.933 3.091 3.400 3.520 3.240 3.917 3.210 3.143 3.773 L4 3.433 3.210 3.322 2.933 3.155 3.653 3.700 3.477 4.167 3.337 2.837 3.793 L5 3.390 3.183 3.287 3.170 3.213 3.630 3.683 3.613 4.307 3.280 3.287 3.703 L6 3.613 3.513 3.563 3.243 3.440 3.683 3.930 3.810 4.253 3.250 3.240 3.710 L1 2.423 2.467 2.445 2.393 2.435 2.803 2.923 2.550 3.233 2.233 2.480 2.683 L2 2.560 2.220 2.390 2.347 2.319 3.043 3.163 3.010 3.210 2.413 2.750 2.820 L3 2.377 2.177 2.277 2.370 2.274 2.683 3.010 2.617 3.340 2.400 2.467 2.830 L4 2.410 1.983 2.197 2.017 2.066 2.963 2.517 2.367 3.270 2.220 2.310 2.567 L5 2.567 2.400 2.483 2.267 2.383 2.593 2.797 2.677 3.353 2.430 2.400 2.850 L6 2.523 2.287 2.405 2.550 2.414 2.823 2.790 2.800 3.390 2.580 2.617 2.810 CA L1 3.133 3.167 3.150 2.737 3.018 3.540 3.693 3.743 3.927 3.090 3.070 3.583 L2 3.237 3.210 3.223 2.847 3.093 3.513 3.603 3.743 4.140 3.307 3.303 3.710 L3 3.257 3.343 3.300 3.153 3.266 3.597 3.807 3.280 4.133 3.477 3.527 3.803 L4 3.373 3.433 3.403 3.200 3.346 3.877 3.947 3.823 4.487 3.110 3.500 4.000 L5 3.480 3.880 3.680 3.610 3.723 3.937 4.193 4.027 4.603 3.603 3.863 4.373 L6 3.603 3.920 3.762 3.620 3.767 3.967 4.120 4.050 4.513 3.990 3.860 4.147 Interaction between these two factors was insignifi cant (F=0.042; p>0.9999) since there was a same trend of L , L and L in all lumbar vertebrae. Namely, L was D M V V the smallest while L was the largest in all vertebrae. The bodies of the L4 and L5 vertebrae had the greatest length, thus Tukey’s HSD test (main effect of the number of vertebrae) showed signifi cant differences in the lengths of both L4 and L5 compared to those of L1, L2 and L6. The body of L1 had the smallest length and in addition to L4 and L5, it differed signifi cantly from L3 (Figure 2A). Tukey’s HSD test (main effect of the level of measurement) also showed that both dorsal length (L ) and medial length (L ) were signifi cantly different, i.e. greater compared to the ventral length (L ), with no signifi cant differences between L and L , although L had the greatest V D M D mean value. Pearson’s correlation coeffi cient showed a signifi cant positive correlation between the guinea pigs’ body weight and the lengths of all lumbar vertebrae in all three levels of measurement (Table 1A). The average correlation coeffi cient was 0.870. This relationship between the length of lumbar vertebrae and body weight was confi rmed by regression analysis. The slopes of all regression lines (for all vertebrae in every level of measurement) were signifi cantly different from zero, while there was no signifi cant difference between the slopes of the different lines (Figure 2B, Supplementary Table S2). 61 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Table 1. Pearson’s correlation coeffi cient (r) of the body weight and morphometric parameters of lumbar vertebrae. L1 L2 L3 L4 L5 L6 0.882 0.869 0.882 0.887 0.897 0.864 *** *** *** *** **** *** 0.839 0.877 0.884 0.871 0.879 0.842 A) length of vertebrae body L *** *** *** *** *** *** 0.852 0.824 0.851 0.890 0.901 0.865 *** *** *** *** **** *** 0.506 0.631 0.584 0.292 0.456 0.141 CR n.s. * * n.s. n.s. n.s. 0.277 0.536 0.541 0.414 0.415 0.580 B) height of vertebrae body H n.s. n.s. n.s. n.s. n.s. * 0.455 0.592 0.603 0.418 0.547 0.653 CA n.s. * * n.s. n.s. * 0.797 0.809 0.746 0.672 0.735 0.594 C) depth of spinal canal D ** ** ** * ** * Length of guinea pig lumbar vertebrae bodies (L1 to L6) at the dorsal (LD), medial (LM) and ventral (LV) levels; height of guinea pig lumbar vertebrae bodies at the cranial (HCR), medial (HM) and caudal (HCA) levels; depth (D) of the spinal canal of lumbar vertebrae. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; n.s. – nonsignifi cant. Height of lumbar verte bral bodies Two-way ANOVA showed a signifi cant difference in the heights of different lumbar vertebrae (F=7.593; p<0.0001), a signifi cant difference between the different levels of measurement, namely cranial (H ), medial (H ) and caudal (H ) (F=189.7; p<0.0001), CR M CA as well as a signifi cant interaction between these two factors (F=2.439; p<0.01) (Figure 3A, Supplementary Tables S1 and S3). L5 and L6 had the greatest height. Tukey’s HSD test (main effect of the number of vertebrae) showed a signifi cant difference in the heights of both L5 and L6 compared to L1 and L2, while that of L6 was signifi cantly higher compared to L3 and L4 as well. Tukey’s HSD test (main effect of the level of measurement) showed signifi cant differences between all three levels of measurement, with H being the greatest and H the smallest. Additionally, height at the level of CA M both cranial and caudal epiphyses showed a trend of increase from L1 to L6. Namely, at the level of the cranial epiphysis, the post-hoc test showed that L6 was signifi cantly greater compared to both L1 and L2, while height at the level of the caudal epiphysis was signifi cantly greater in L5 and L6 compared to L1, L2 and L3 (Figure 3A). On the other hand, there was neither trend nor any signifi cant difference at the medial level. L4 had the smallest average H , which is consistent with the signifi cant two-way ANOVA interaction. In addition, L4 had the greatest difference between H and H , CR M and together with L6 the greatest difference between H and H (Figure 3A). Unlike CA M lumbar vertebral length, height was not as strongly correlated with body mass/weight 62 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect Figure 3. (A) Height of guinea pig lumbar vertebrae bodies in the cranial, medial and caudal levels. (B) Linear regression of lumbar vertebrae body heights as the dependent variable on body weight as the independent variable. HCR ‒ height in the cranial part of the vertebral body; HM ‒ height in the medial part of the vertebral body; HCA ‒ height in the caudal part of the vertebral body; ** p<0.01; *** p<0.001; **** p<0.0001; n.s. – nonsignifi cant. 63 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 (Table 1B). Pearson’s correlation coeffi cient showed a signifi cant positive correlation between guinea pig body weight and the height of the cranial and caudal epiphyses of L2 and L3 as well as the caudal epiphysis and medial height of L6. Correlation coeffi cients for the listed parameters were in the range from 0.580 to 0.631, with an average correlation coeffi cient of only 0.480. Similarly, regression analysis (Figure 3B, Supplementary Table S3) showed that only the slopes of the regression lines for the cranial and caudal epiphyses of L2 and L3 as well as the caudal epiphysis and medial height of L6 were signifi cantly different from zero, while there were no signifi cant differences between the slopes of different lines. Supplementary table S3: Depth of the spinal canal (in millimeters). L1 2.127 2.270 2.310 2.393 2.063 2.377 2.483 2.997 2.353 2.790 3.160 2.750 L2 2.130 2.437 2.360 2.493 2.210 2.467 2.507 3.080 2.503 3.007 3.173 2.723 L3 2.433 2.667 2.547 2.603 2.490 2.683 2.680 3.283 2.533 3.180 3.500 2.937 L4 2.410 2.820 2.867 2.973 2.557 2.727 2.997 3.507 2.597 3.463 3.500 3.107 L5 2.217 2.507 2.413 2.947 2.230 3.000 2.817 3.217 2.520 3.340 3.280 2.937 L6 1.623 1.783 1.697 2.193 1.670 2.243 1.980 2.320 1.867 2.840 1.937 2.083 Depth of the spinal canal Similar to the length of lumbar vertebrae, the depth of the spinal canal was characterized by greater variability, with the greatest depth in L4 and the smallest in L6. One-way ANOVA showed signifi cant differences (F=10.2; p<0.0001). Tukey’s HSD test showed that the depth of the spinal canal of L4 was signifi cantly greater compared to L1 and L6. In addition, the spinal canal of L6 was signifi cantly smaller compared to all the other lumbar vertebrae (Figure 4A, Supplementary Tables S1 and S4). Pearson’s correlation coeffi cient showed a signifi cant positive correlation between the guinea pigs’ body weight and the depth of the spinal canal of all six lumbar vertebrae (Table 1C). The average correlation coeffi cient was 0.726. This relationship between depth of lumbar canal and body mass/weight was confi rmed by regression analysis. The slopes of all regression lines (for all vertebrae) were signifi cantly different from zero, while there was no signifi cant difference between the slopes of the different lines (Figure 4B). DISCUSSION Regulation of bone growth is a complex process that depends on genetic factors, vascularization [17], the infl uence of hormones [18, 19], as well as biomechanical factors [20]. The infl uence of mechanical force on bone growth is often considered through Hueter-Volkmann’s law, which indicates that an increase in pressure in the epiphyseal growth zones has an inhibitory effect, as opposed to distension, which promotes longitudinal bone growth [21]. In studies performed on rats, a decrease in 64 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect Figure 4. (A) Depth of the spinal canal of guinea pig lumbar vertebrae. (B) Linear regression of depth of the spinal canal of lumbar vertebrae as the dependent variable on body weight as the independent variable. DSC ‒ depth of the spinal canal; * p<0.05; ** p<0.01; **** p<0.0001. the growth of the forelimbs was observed with increased axial load [22], while the body length of the caudal vertebrae recorded an increase of 14% in distraction, i.e. a decrease of 22% in increasing axial pressure [23]. Nevertheless, the biomechanical impact on bone growth and morphology is much more complex and there are a lot of inconsistent data in the available literature [24]. 65 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Although the mechanical modulation of longitudinal bone growth by compressive forces is best known, the effects of torsion and bending on longitudinal, rotational and angular bone development should not be ignored [25]. The infl uence of spinal fl exion on the morphology of the vertebral body was examined by [26]. They suggested that seals and dolphins have round vertebral bodies due to pronounced lateral and sagittal fl exion, while in tetrapedal organisms, due to predominant sagittal fl exion, the vertebral body has an irregular quadrangular shape. The results of our study showed that the body length of the lumbar vertebrae of guinea pigs differs in the dorsal, medial and ventral areas, the dorsal length being the largest and the ventral the smallest. Hueter-Volkmann’s law, which describes the infl uence of force on bone growth, points out that during skeletal development bone growth is relatively retarded in places of increased mechanical compression and relatively accelerated in places of reduced load [27]. This may explain the irregular trapezoidal geometry of the bodies of the lumbar vertebrae of guinea pigs, which due to sagittal fl exion and arched lumbar spine, exert the greatest pressure in the cranioventral and caudoventral parts of the bodies of two adjacent vertebrae, which could lead to the earlier closure of the ventral epiphyseal growth zone with unhindered development of the proximal part of the vertebral body. The shape and size of the vertebrae, as well as their position inside the spinal column, should enable an even distribution and transfer of forces through the central part of the vertebral body. In bipedal organisms, pressure on the spinal column increases in a caudal direction, which is accompanied by a proportional increase in the dimensions of the vertebral body [28], so that the last lumbar vertebra has the greatest length, width and height [4]. Takahashi et al. also showed that in humans the greatest load is exerted in the lumbar region of the spine, and that disc protrusions are most common between L4 and L5 [29]. According to the results of our study, guinea pigs did not exhibit a proportional increase in all the examined morphometric parameters of the lumbar vertebrae in the craniocaudal direction, as occurs in humans, but the body lengths of L4 and L5 were signifi cantly longer than those of L1, L2 and L6. On the other hand, the heights of the lumbar vertebrae generally showed an increase in the caudal direction, so that the heights of the cranial epiphyses in L1 and L2 are signifi cantly lower than in L6, and the caudal epiphyses of L1, L2 and L3 have signifi cantly lower values than L5 and L6. The heights measured in the central part of the body are uniform and do not show signifi cant deviations between the vertebrae. However, they are signifi cantly smaller in relation to the height of the cranial and caudal epiphyses, as a result of which the ventral outline of the vertebrate body of guinea pigs has a slight concavity. The concavity is the most pronounced in L4, which is characterized by the smallest medial height and the greatest difference in medial height versus the height of the cranial and caudal epiphyses. The relationship between the length and height of the vertebral body in tetrapedal organisms is correlated with a different mechanical load on the lumbar spine and the transmission of force through the vertebral body. Therefore, differences observed in 66 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect heights and the concave appearance of the ventral outline of the vertebral body in guinea pigs can be explained as an adaptive response of the bone to bending forces that can occur in these animals due to lordosis of the lumbar spine. Martin and Burr indicated that when the bone is bent, fl uid is pushed through the canalicular bone system against the action of force and performs a mechanical stimulus on the cells, which ultimately results in bone adjustment to mechanical load and the appearance of curvature of the periosteal and endocortical bones [30]. The basic function of the spinal column is to provide mechanical stability and protect the spinal cord. The vertebral body, as the bearer of the greatest load, together with the vertebral arch, form the spinal canal and protect the spinal cord from injuries. However, the diameter of the spinal canal is not uniform along its entire length and in humans its width increases from the fi rst to the last lumbar vertebra, while the depth is lowest at the L3 level and increases in a caudal direction [31]. Thus, in humans, there is a correlation between the diameter of the spinal canal and the place of greatest load on the spine, which can be considered a remarkable protective mechanism that aims to prevent pressure on the spinal cord that could occur when a higher mechanical force is applied. The results of our study show that in guinea pigs, the greatest depth of the spinal canal was measured at the level of L4, and the smallest in L6. CONCLUSION The results of our study indicate that in guinea pigs there is no linear increase in the values of morphometric parameters of the lumbar vertebrae in the direction from the fi rst to the last, as is the case in humans. In guinea pigs, L4 and L5 were observed to have the greatest body lengths, while the ventral side of the body of L4 was the most concave. Additionally, the depth of the spinal canal was greatest at the level of L4. This atypical morphology of the lumbar vertebrae leads to the conclusion that the direction of force transmission through the spinal column of tetrapedal organisms differs from that in bipedal organisms, and that the center of gravity of the load is shifted in a cranial direction. Also, the atypical morphology of L4 could indicate that this vertebra has the role of “shock absorber” and that it suffers the greatest load. However, it must be remembered that this conclusion was made on the basis of vertebral body morphometry alone, while the biomechanics of the spine are infl uenced by numerous other factors such as bone structure (direction of provision trabeculae and density), the characteristics of facet joints, intervertebral discs and associated soft tissue structures. Acknowledgments This work was predominantly funded by authors and to a lesser extent by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. 451-03-68/2022-14/200007). 67 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Author’s contributions MM, SK, and MLM conceived of the study, participated in its design and coordinated the work. Morphometric measurements were performed by MM, SK, and AT. NT, MM, and SK performed the statistical analysis. MM, SK, and AT wrote the manuscript in consultation with MLM and NT. All authors read and approved the fi nal manuscript. Declaration of confl icting of interests The author(s) declared no potential confl icts of interest with respect to the research, authorship, and/or publication of this article. REFERENCES 1. Goh S, Tan C, Price RI, Edmondston SJ, Song S, Davis S & Singer KP: Infl uence of age and gender on thoracic vertebral body shape and disc degeneration: An MR investigation of 169 cases. J Anat, 2013, 197: 647-657. 2. Frost BA, Camarero-Espinosa S, Foster EJ: Materials for the Spine: Anatomy, Problems and Solutions. Materials, 2019, 12(2): 253. 3. Yavropoulou MP, Yovos JG: The molecular basis of bone mechanotransduction. JMNI, 2016, 16(3): 221-236. 4. Tan SH, Teo EC & Chua HC: Quantitative three-dimensional anatomy of cervical, thoracic and lumbar vertebrae of Chinese Singaporeans. Eur Spine J, 2004, 13(2): 137-146. 5. Guerner JN, Erulkar JS, Patel TC, Panjab MM: Biomechanical evaluation of the New Zeland white rabbit lumbar spine: a physiologic characteristic. Eur Spine J, 2000, 9(3): 250- 6. 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J Bone Joint Surg, 1980, 62-B(2): 230-237. 26. Boszczyk BM, Boszczyk AA, Putz R: Comparative and functional anatomy of the mammalian lumbar spine. Anat Rec, 2011, 264(2): 157-68. 27. Hunter C: Anatomische Studien and den Extremitatengelenken Neugeborner und Erwachsener. Virchows Arch, 1862, 25: 572-599. 28. Daggfeldt K, Thorstensson A: The mechanics of back-extensor torque production about the lumbar spine. J Biomech, 2003, 36(6): 815-825. 29. Takahashi I, Kikuchi S, Sato K: Mechanical load of the lumbar spine during forward bending motion of the trunk – a biomechanical study. Spine, 2006, 31(1): 18-23. 30. Martin RB, Burr DB: The Structure, Function and Adaptation of Compact Bone (Reven Press, New York, USA), 1989, 275. 31. Griffi th JF, Huang J, Law SW, Xiao F, Leung JCS, Wang D, Shi L: Population reference for development lumbar spinal canal size. Quant Imag Med Surg, 2016, 6(6): 671-679. 69 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 RADIOLOŠKA ISPITIVANJA MORFOLOGIJE LUMBALNIH PRŠLJENOVA ZAMORACA (CAVIA PORCELLUS) – BIOMEHANIČKI ASPEKT Marko Jumake MITROVIĆ, Sara KITANOVIĆ, Nikola TATALOVIĆ, Anastasija TODOROVIĆ, Mirjana LAZAREVIĆ MACANOVIĆ Brojne studije su zasnovane na upotrebi animalnih modela, ipak, kod bipedalnih i te- trapedalnih organizama postoje značajne razlike u biomehanici kičmenog stuba, koje mogu značajno narušiti kvalitet i primenjivost dobijenih rezultata. Cilj ovog ispitivanja je da se dobiju osnovni podaci o morfometrijskim parametrima lumbalnih pršljenova zamoraca, čijom analizom će se ukazati na mesto najvećeg mehaničkog opterećenja. Morfometrijsko ispitivanje lumbalnih pršljenova zamoraca je sprovedeno na rendgen- skim snimcima 12 jedinki, sa jednakim brojem mužjaka i ženki. Rezultati ispitivanja pokazuju da lumbalni pršljenovi zamoraca imaju nepravilnu trapezoidnu geometriju i da su izmerene dužine L4 i L5 najveće. Parametri visine određivani su u medijalnom nivou i pokazali su da L4 ima najkonkavnije telo. Štaviše, L4 je imao najveću dubinu kičmenog kanala na istom nivou merenja. Shodno tome, kod zamoraca je najveće op- terećenje u L4 regiji za razliku od ljudi, gde se usled aksijalnog opterećenja kičmenog stuba najveći pritisak ostvaruje na poslednjim lumbalnim pršljenovima. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta veterinaria de Gruyter

Radiological Investigation of Guinea Pig (Cavia porcellus) Lumbar Vertebral Morphology ‒ A Biomechanical Aspect

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de Gruyter
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© 2023 Marko Jumake Mitrović et al., published by Sciendo
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1820-7448
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DOI
10.2478/acve-2023-0005
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Abstract

Acta Veterinaria-Beograd 2023, 73 (1), 55-70 UDK: 599.324.7:591.471.35 DOI: 10.2478/acve-2023-0005 Research article RADIOLOGICAL INVESTIGATION OF GUINEA PIG (CAVIA PORCELLUS) LUMBAR VERTEBRAL MORPHOLOGY ‒ A BIOMECHANICAL ASPECT 1 1 2 Marko Jumake MITROVIĆ *, Sara KITANOVIĆ , Nikola TATALOVIĆ , 1 1 Anastasija TODOROVIĆ , Mirjana LAZAREVIĆ MACANOVIĆ University of Belgrade, Faculty of Veterinary Medicine, Department of Radiology and Radiation Hygiene, Bulevar oslobođenja 18, Belgrade, Serbia; University of Belgrade, Institute for Biological Research “Siniša Stanković” ‒ National Institute of Republic of Serbia, Department of Physiology, Bulevar Despota Stefana, Belgrade, Serbia (Received 17 September, Accepted 22 December 2022) Numerous studies are based on the use of animal models; however, in bipedal and tetrapedal organisms there are signifi cant differences in the biomechanics of the spinal column, which can signifi cantly impair the quality and applicability of the results obtained. The aim of this study is to obtain basic data on the morphometric parameters of guinea pig lumbar vertebrae, the analysis of which will indicate the location of the biggest mechanical load. The lumbar vertebra morphometry test was performed by means of X-ray imageing obtained from 12 guinea pigs, with equal numbers of males and females. The results of investigations show that guinea pig lumbar vertebrae have an irregular trapezoid geometry and that the measured body lengths of L4 and L5 are the largest. The height parameters determined in the medial level showed that L4 had the most concave body. Moreover, L4 had the greatest depth of the spinal canal at the same measurement level. Consequently, in guinea pigs, the greatest load is in the L4 region, unlike in humans, where, due to the axial load of the spinal column, the highest pressure is exerted on the last lumbar vertebrae. Keywords: biomechanics, guinea pig, lumbar spine, morphometry, radiology INTRODUCTION Knowledge of biomechanics enables a better understanding of the role of bones and associated soft tissues in stabilizing the spine, as well as an understanding of the destabilizing effects that can occur due to the appearance of tumors, traumatic and degenerative damages [1]. In a functional sense, bone and soft tissue structures are closely connected and their mutual action achieves the stability of the spine and its mobility in all three axes [2]. *Corresponding author: e-mail: markom@vet.bg.ac.rs 55 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 In humans, who are bipedal organisms, the thoracic part of the spine has limited mobility because ribs are attached to the vertebrae at one end and to the sternum at the other end. Unlike the thoracic segment, the mobility of the lumbar spine is signifi cantly higher and at the same time, it bears the greatest axial load [2]. It is known that bones adapt to the action of mechanical forces by changing their morphology [3], so the size of the thoracic vertebrae in humans increases from the fi rst to the last [1], and this trend continues distally so that the last lumbar vertebra has the greatest length, height and width [4]. Considering the high incidence of various pathological conditions in the spinal column of humans, which predominantly affect the lumbar region, numerous studies are aimed at fi nding an adequate biomechanical model to examine the static-mechanical relationships of lumbar vertebrae. In the available literature, there are various fi ndings of different morphometric and/or biomechanics examinations of the spinal column performed on rabbits [5], dogs [6], pigs [7], sheep [8] and cattle [9,10]; however, the search for an optimal animal model is still ongoing. It should be borne in mind that there are signifi cant differences in the biomechanics of the spinal columns of bipedal and tetrapedal organisms (Supplementary Figure S1), which can signifi cantly impair the quality and applicability of the results obtained. Supplementary Figure S1: Presentation of the morphometry of the spinal column in different species of mammals. Guinea pigs (Cavia porcellus) are a herbivorous species of rodents from the family Caviidae which due to their low body weight, short reproductive cycle, calm temperament, as well as anatomical and physiological characteristics, are increasingly used as models in various studies [11]. In neurology and orthopedics, guinea pigs have been used to examine ischemic lesions in the spinal cord [12], and also for the study of spontaneous and induced osteoarthritis [13]. The spinal column of rodents has a total of 26 presacral vertebrae, composed of 7 cervical and 19 thoracolumbar vertebrae [14]. The cervical spine region of mammals is characterized by the smallest variations 56 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect in the number of vertebrae, which is usually attributed to the pleiotropic function of Hox genes. In addition, variations in the number of cervical vertebrae are associated with an increased risk of prenatal mortality and neonatal cancer [14,15]. On the other side, the thoracolumbar region of the spine of guinea pigs shows greater variability, and in most individuals, it is composed of 13 thoracic and 6 lumbar vertebrae [16]. The aim of this study was to obtain data on the morphometric parameters of the lumbar vertebrae of guinea pigs and to view them from the aspect of biomechanics in order to obtain information on the mechanical load on the lumbar spine of these animals. MATERIAL AND METHODS Animals The examination was performed on 12 guinea pigs (Cavia porcellus), with an equal number of males (6) and females (6), body weight 380-800 g. All morphometric examinations of the lumbar vertebrae were performed on X-rays (permission no. 323-07-0850112019-05 issued by the Veterinary Administration of the Ministry of Agriculture and Environmental Protection of the Republic of Serbia). Experimental procedures were performed in compliance with Directive 2010/63/EU on the protection of animals used for experimental and other scientifi c purposes. Preparation for radiological examination In order to minimize the guinea pigs’ movements and to obtain images of optimal quality for analysis, all animals were anesthetized following a three-hour deprivation of food and water. Animals were anesthetized using ketamine hydrochloride (Ketamidor 10%, Richter Pharma, Austria) in a dose of 60 mg/kg body weight, with xylazine hydrochloride premedication (Xylased, Bioveta, Czech Republic) in a dose of 4 mg/ kg. The application was performed subcutaneously, using a 25 G-diameter injection needle. Radiological examination All radiographs were performed using a ZooMax Gold X-ray machine (Control-X Medical, Hungary). The animals were placed in a plexiglass basket, and morphometric examinations of various parameters of the lumbar vertebrae were performed only on images made in lateral recumbency in the laterolateral projection (Figure 1A) because in the ventrodorsal projection it was not possible to accurately measure the width of the vertebral body due to summation of shadows of gastrointestinal contents and bony outlines of vertebrae. The X-rays were performed at a focus fi lm distance of 90 cm, using a voltage of 44 kVp, while the exposure was 5 mAs. 57 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Morphometric examinations of lumbar vertebrae Morphometric examinations of all six lumbar vertebrae (L1-6) of guinea pigs were performed in the RadiAnt DICOM Viewer program by three examiners. The length of vertebral bodies was measured at three different levels: dorsal (L ), medial (L ) and D M ventral (L ), as well as the height: cranial (H ), medial (H ) and caudal (H ). The V CR M CA depth of the spinal canal was measured in the central part of each examined vertebra (Figure 1B-D). Figure 1. Lateral recumbency of guinea pig in a plexiglass basket (A) and morphometric examinations of lumbar vertebrae: (B) measuring the length in the dorsal (LD), medial (LM) and ventral (LV) parts of the vertebral body; (C) measuring height in the cranial (HCR), medial (HM) and caudal (HCA) parts of the body; (D) measuring the depth of the spinal canal (DSC). Data analysis and statistical procedures Morphometric parameters of lumbar vertebrae (length and height of vertebral bodies at three different levels and depth of the spinal canal) are presented as mean ± standard deviation (SD) in millimeters. All individual values are presented in the Supplementary Material. The sample size (n) was 12. Differences in the length and height of vertebrae bodies in all three levels were tested by factorial analysis of variance (two-way ANOVA) with the number of vertebrae and the level of measurement as factors, and post hoc compared by Tukey’s honest signifi cant difference (HSD) test. Differences in depth of the spinal canal were tested by single factor analysis of variance (one-way ANOVA) 58 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect and post hoc compared by Tukey’s HSD test. Correlations between body weight and the morphometric parameters of lumbar vertebrae were examined using Pearson’s correlation coeffi cient as well as linear regression of morphometric parameters as the dependent variable on body weight as the independent variable. Deviations in linear regression curve slopes from zero were tested by the F-test. Comparisons of linear regression curve slopes were also performed using the F-test. Statistical signifi cance of the correlation coeffi cient was tested using Student’s t-test. The signifi cance level was 0.05. All analyses were conducted using GraphPad Prism 8 (GraphPad Software Inc.). RESULTS Length of lumbar vertebral bodies Two-way ANOVA showed a signifi cant difference in the lengths of different lumbar vertebrae (F=15.4; p<0.0001) as well as a signifi cant difference between the different levels of measurement, namely dorsal (L ), medial (L ) and ventral (L ) (F=8.754; D M V p<0.001) (Figure 2A, Supplementary Tables S 1 and S2). Supplementary table S1: Length of lumbar vertebral bodies (in millimeters); L – length in the dorsal level; L – length in the medial level; L ‒ length in the ventral level. M V L1 6.567 6.870 6.903 7.380 7.320 7.133 8.260 9.177 8.070 8.527 9.133 8.320 L2 7.160 7.390 7.460 8.170 8.000 7.843 8.980 10.167 8.823 9.383 9.870 8.963 L3 7.557 7.927 7.963 8.737 8.420 8.417 9.573 10.733 9.400 10.100 10.567 9.557 L4 8.047 8.263 8.293 9.217 8.893 8.893 10.000 11.233 9.960 10.633 11.000 9.990 L5 7.797 8.073 8.220 9.303 8.670 9.403 9.700 11.233 10.100 11.133 11.100 9.777 L6 6.787 7.067 7.073 8.230 7.767 8.840 8.387 10.097 8.863 10.667 10.153 8.480 L1 6.317 6.417 6.450 6.857 7.320 6.690 7.743 8.830 7.560 8.167 8.847 7.657 L2 6.820 6.977 7.137 7.777 7.820 7.507 8.517 9.643 8.473 9.023 9.600 8.540 L3 7.437 7.627 7.617 8.253 8.097 8.173 9.120 10.233 9.020 9.710 10.247 9.023 L4 7.780 7.993 8.103 8.897 8.793 8.573 9.620 10.833 9.577 10.267 10.733 9.507 L5 7.690 7.863 8.003 9.170 8.590 9.117 9.497 10.967 9.803 10.933 10.900 9.423 L6 6.797 7.000 7.020 8.263 7.720 8.630 8.407 10.100 8.670 10.233 9.947 8.273 L1 5.597 6.003 5.943 6.537 6.457 6.247 7.587 8.560 7.097 7.913 8.573 7.337 L2 6.357 6.483 6.693 7.477 7.090 6.650 8.350 9.210 7.900 8.473 9.143 8.017 L3 7.170 7.440 7.263 8.067 7.563 7.427 8.883 9.743 8.830 8.903 9.897 8.833 L4 7.393 7.810 7.797 8.457 8.010 8.003 9.173 10.367 9.400 10.067 10.600 9.267 L5 7.373 7.397 7.390 8.590 7.917 8.690 8.783 10.300 9.180 10.467 10.600 9.133 L6 6.060 6.507 6.610 7.450 7.087 8.087 7.883 9.600 7.890 9.757 9.587 7.973 59 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Figure 2. (A) Length of guinea pig lumbar vertebrae bodies in dorsal, medial and ventral levels. (B) Linear regression of lumbar vertebrae bodies lengths as the dependent variable on body weight as the independent variable. LD ‒ length in the dorsal part of the vertebral body; LM ‒ length in the medial part of the vertebral body; LV ‒ length in the ventral part of the vertebral body; p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; n.s. – nonsignifi cant. 60 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect Supplementary table S2: Height of lumbar vertebral bodies (in millimeters); H – height CR in the level of cranial epiphysis; H – height in the medial level; H – height in the level of M CA caudal epiphysis. CR L1 3.003 2.950 2.977 2.573 2.833 3.473 3.483 3.100 3.643 2.973 3.023 3.583 L2 2.887 3.003 2.945 2.750 2.899 3.327 3.307 3.400 3.697 3.173 2.930 3.500 L3 2.910 3.257 3.083 2.933 3.091 3.400 3.520 3.240 3.917 3.210 3.143 3.773 L4 3.433 3.210 3.322 2.933 3.155 3.653 3.700 3.477 4.167 3.337 2.837 3.793 L5 3.390 3.183 3.287 3.170 3.213 3.630 3.683 3.613 4.307 3.280 3.287 3.703 L6 3.613 3.513 3.563 3.243 3.440 3.683 3.930 3.810 4.253 3.250 3.240 3.710 L1 2.423 2.467 2.445 2.393 2.435 2.803 2.923 2.550 3.233 2.233 2.480 2.683 L2 2.560 2.220 2.390 2.347 2.319 3.043 3.163 3.010 3.210 2.413 2.750 2.820 L3 2.377 2.177 2.277 2.370 2.274 2.683 3.010 2.617 3.340 2.400 2.467 2.830 L4 2.410 1.983 2.197 2.017 2.066 2.963 2.517 2.367 3.270 2.220 2.310 2.567 L5 2.567 2.400 2.483 2.267 2.383 2.593 2.797 2.677 3.353 2.430 2.400 2.850 L6 2.523 2.287 2.405 2.550 2.414 2.823 2.790 2.800 3.390 2.580 2.617 2.810 CA L1 3.133 3.167 3.150 2.737 3.018 3.540 3.693 3.743 3.927 3.090 3.070 3.583 L2 3.237 3.210 3.223 2.847 3.093 3.513 3.603 3.743 4.140 3.307 3.303 3.710 L3 3.257 3.343 3.300 3.153 3.266 3.597 3.807 3.280 4.133 3.477 3.527 3.803 L4 3.373 3.433 3.403 3.200 3.346 3.877 3.947 3.823 4.487 3.110 3.500 4.000 L5 3.480 3.880 3.680 3.610 3.723 3.937 4.193 4.027 4.603 3.603 3.863 4.373 L6 3.603 3.920 3.762 3.620 3.767 3.967 4.120 4.050 4.513 3.990 3.860 4.147 Interaction between these two factors was insignifi cant (F=0.042; p>0.9999) since there was a same trend of L , L and L in all lumbar vertebrae. Namely, L was D M V V the smallest while L was the largest in all vertebrae. The bodies of the L4 and L5 vertebrae had the greatest length, thus Tukey’s HSD test (main effect of the number of vertebrae) showed signifi cant differences in the lengths of both L4 and L5 compared to those of L1, L2 and L6. The body of L1 had the smallest length and in addition to L4 and L5, it differed signifi cantly from L3 (Figure 2A). Tukey’s HSD test (main effect of the level of measurement) also showed that both dorsal length (L ) and medial length (L ) were signifi cantly different, i.e. greater compared to the ventral length (L ), with no signifi cant differences between L and L , although L had the greatest V D M D mean value. Pearson’s correlation coeffi cient showed a signifi cant positive correlation between the guinea pigs’ body weight and the lengths of all lumbar vertebrae in all three levels of measurement (Table 1A). The average correlation coeffi cient was 0.870. This relationship between the length of lumbar vertebrae and body weight was confi rmed by regression analysis. The slopes of all regression lines (for all vertebrae in every level of measurement) were signifi cantly different from zero, while there was no signifi cant difference between the slopes of the different lines (Figure 2B, Supplementary Table S2). 61 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Table 1. Pearson’s correlation coeffi cient (r) of the body weight and morphometric parameters of lumbar vertebrae. L1 L2 L3 L4 L5 L6 0.882 0.869 0.882 0.887 0.897 0.864 *** *** *** *** **** *** 0.839 0.877 0.884 0.871 0.879 0.842 A) length of vertebrae body L *** *** *** *** *** *** 0.852 0.824 0.851 0.890 0.901 0.865 *** *** *** *** **** *** 0.506 0.631 0.584 0.292 0.456 0.141 CR n.s. * * n.s. n.s. n.s. 0.277 0.536 0.541 0.414 0.415 0.580 B) height of vertebrae body H n.s. n.s. n.s. n.s. n.s. * 0.455 0.592 0.603 0.418 0.547 0.653 CA n.s. * * n.s. n.s. * 0.797 0.809 0.746 0.672 0.735 0.594 C) depth of spinal canal D ** ** ** * ** * Length of guinea pig lumbar vertebrae bodies (L1 to L6) at the dorsal (LD), medial (LM) and ventral (LV) levels; height of guinea pig lumbar vertebrae bodies at the cranial (HCR), medial (HM) and caudal (HCA) levels; depth (D) of the spinal canal of lumbar vertebrae. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; n.s. – nonsignifi cant. Height of lumbar verte bral bodies Two-way ANOVA showed a signifi cant difference in the heights of different lumbar vertebrae (F=7.593; p<0.0001), a signifi cant difference between the different levels of measurement, namely cranial (H ), medial (H ) and caudal (H ) (F=189.7; p<0.0001), CR M CA as well as a signifi cant interaction between these two factors (F=2.439; p<0.01) (Figure 3A, Supplementary Tables S1 and S3). L5 and L6 had the greatest height. Tukey’s HSD test (main effect of the number of vertebrae) showed a signifi cant difference in the heights of both L5 and L6 compared to L1 and L2, while that of L6 was signifi cantly higher compared to L3 and L4 as well. Tukey’s HSD test (main effect of the level of measurement) showed signifi cant differences between all three levels of measurement, with H being the greatest and H the smallest. Additionally, height at the level of CA M both cranial and caudal epiphyses showed a trend of increase from L1 to L6. Namely, at the level of the cranial epiphysis, the post-hoc test showed that L6 was signifi cantly greater compared to both L1 and L2, while height at the level of the caudal epiphysis was signifi cantly greater in L5 and L6 compared to L1, L2 and L3 (Figure 3A). On the other hand, there was neither trend nor any signifi cant difference at the medial level. L4 had the smallest average H , which is consistent with the signifi cant two-way ANOVA interaction. In addition, L4 had the greatest difference between H and H , CR M and together with L6 the greatest difference between H and H (Figure 3A). Unlike CA M lumbar vertebral length, height was not as strongly correlated with body mass/weight 62 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect Figure 3. (A) Height of guinea pig lumbar vertebrae bodies in the cranial, medial and caudal levels. (B) Linear regression of lumbar vertebrae body heights as the dependent variable on body weight as the independent variable. HCR ‒ height in the cranial part of the vertebral body; HM ‒ height in the medial part of the vertebral body; HCA ‒ height in the caudal part of the vertebral body; ** p<0.01; *** p<0.001; **** p<0.0001; n.s. – nonsignifi cant. 63 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 (Table 1B). Pearson’s correlation coeffi cient showed a signifi cant positive correlation between guinea pig body weight and the height of the cranial and caudal epiphyses of L2 and L3 as well as the caudal epiphysis and medial height of L6. Correlation coeffi cients for the listed parameters were in the range from 0.580 to 0.631, with an average correlation coeffi cient of only 0.480. Similarly, regression analysis (Figure 3B, Supplementary Table S3) showed that only the slopes of the regression lines for the cranial and caudal epiphyses of L2 and L3 as well as the caudal epiphysis and medial height of L6 were signifi cantly different from zero, while there were no signifi cant differences between the slopes of different lines. Supplementary table S3: Depth of the spinal canal (in millimeters). L1 2.127 2.270 2.310 2.393 2.063 2.377 2.483 2.997 2.353 2.790 3.160 2.750 L2 2.130 2.437 2.360 2.493 2.210 2.467 2.507 3.080 2.503 3.007 3.173 2.723 L3 2.433 2.667 2.547 2.603 2.490 2.683 2.680 3.283 2.533 3.180 3.500 2.937 L4 2.410 2.820 2.867 2.973 2.557 2.727 2.997 3.507 2.597 3.463 3.500 3.107 L5 2.217 2.507 2.413 2.947 2.230 3.000 2.817 3.217 2.520 3.340 3.280 2.937 L6 1.623 1.783 1.697 2.193 1.670 2.243 1.980 2.320 1.867 2.840 1.937 2.083 Depth of the spinal canal Similar to the length of lumbar vertebrae, the depth of the spinal canal was characterized by greater variability, with the greatest depth in L4 and the smallest in L6. One-way ANOVA showed signifi cant differences (F=10.2; p<0.0001). Tukey’s HSD test showed that the depth of the spinal canal of L4 was signifi cantly greater compared to L1 and L6. In addition, the spinal canal of L6 was signifi cantly smaller compared to all the other lumbar vertebrae (Figure 4A, Supplementary Tables S1 and S4). Pearson’s correlation coeffi cient showed a signifi cant positive correlation between the guinea pigs’ body weight and the depth of the spinal canal of all six lumbar vertebrae (Table 1C). The average correlation coeffi cient was 0.726. This relationship between depth of lumbar canal and body mass/weight was confi rmed by regression analysis. The slopes of all regression lines (for all vertebrae) were signifi cantly different from zero, while there was no signifi cant difference between the slopes of the different lines (Figure 4B). DISCUSSION Regulation of bone growth is a complex process that depends on genetic factors, vascularization [17], the infl uence of hormones [18, 19], as well as biomechanical factors [20]. The infl uence of mechanical force on bone growth is often considered through Hueter-Volkmann’s law, which indicates that an increase in pressure in the epiphyseal growth zones has an inhibitory effect, as opposed to distension, which promotes longitudinal bone growth [21]. In studies performed on rats, a decrease in 64 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect Figure 4. (A) Depth of the spinal canal of guinea pig lumbar vertebrae. (B) Linear regression of depth of the spinal canal of lumbar vertebrae as the dependent variable on body weight as the independent variable. DSC ‒ depth of the spinal canal; * p<0.05; ** p<0.01; **** p<0.0001. the growth of the forelimbs was observed with increased axial load [22], while the body length of the caudal vertebrae recorded an increase of 14% in distraction, i.e. a decrease of 22% in increasing axial pressure [23]. Nevertheless, the biomechanical impact on bone growth and morphology is much more complex and there are a lot of inconsistent data in the available literature [24]. 65 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Although the mechanical modulation of longitudinal bone growth by compressive forces is best known, the effects of torsion and bending on longitudinal, rotational and angular bone development should not be ignored [25]. The infl uence of spinal fl exion on the morphology of the vertebral body was examined by [26]. They suggested that seals and dolphins have round vertebral bodies due to pronounced lateral and sagittal fl exion, while in tetrapedal organisms, due to predominant sagittal fl exion, the vertebral body has an irregular quadrangular shape. The results of our study showed that the body length of the lumbar vertebrae of guinea pigs differs in the dorsal, medial and ventral areas, the dorsal length being the largest and the ventral the smallest. Hueter-Volkmann’s law, which describes the infl uence of force on bone growth, points out that during skeletal development bone growth is relatively retarded in places of increased mechanical compression and relatively accelerated in places of reduced load [27]. This may explain the irregular trapezoidal geometry of the bodies of the lumbar vertebrae of guinea pigs, which due to sagittal fl exion and arched lumbar spine, exert the greatest pressure in the cranioventral and caudoventral parts of the bodies of two adjacent vertebrae, which could lead to the earlier closure of the ventral epiphyseal growth zone with unhindered development of the proximal part of the vertebral body. The shape and size of the vertebrae, as well as their position inside the spinal column, should enable an even distribution and transfer of forces through the central part of the vertebral body. In bipedal organisms, pressure on the spinal column increases in a caudal direction, which is accompanied by a proportional increase in the dimensions of the vertebral body [28], so that the last lumbar vertebra has the greatest length, width and height [4]. Takahashi et al. also showed that in humans the greatest load is exerted in the lumbar region of the spine, and that disc protrusions are most common between L4 and L5 [29]. According to the results of our study, guinea pigs did not exhibit a proportional increase in all the examined morphometric parameters of the lumbar vertebrae in the craniocaudal direction, as occurs in humans, but the body lengths of L4 and L5 were signifi cantly longer than those of L1, L2 and L6. On the other hand, the heights of the lumbar vertebrae generally showed an increase in the caudal direction, so that the heights of the cranial epiphyses in L1 and L2 are signifi cantly lower than in L6, and the caudal epiphyses of L1, L2 and L3 have signifi cantly lower values than L5 and L6. The heights measured in the central part of the body are uniform and do not show signifi cant deviations between the vertebrae. However, they are signifi cantly smaller in relation to the height of the cranial and caudal epiphyses, as a result of which the ventral outline of the vertebrate body of guinea pigs has a slight concavity. The concavity is the most pronounced in L4, which is characterized by the smallest medial height and the greatest difference in medial height versus the height of the cranial and caudal epiphyses. The relationship between the length and height of the vertebral body in tetrapedal organisms is correlated with a different mechanical load on the lumbar spine and the transmission of force through the vertebral body. Therefore, differences observed in 66 Mitrović et al.: Radiological investigation of guinea pig (Cavia porcellus) lumbar vertebral morphology ‒ a biomechanical aspect heights and the concave appearance of the ventral outline of the vertebral body in guinea pigs can be explained as an adaptive response of the bone to bending forces that can occur in these animals due to lordosis of the lumbar spine. Martin and Burr indicated that when the bone is bent, fl uid is pushed through the canalicular bone system against the action of force and performs a mechanical stimulus on the cells, which ultimately results in bone adjustment to mechanical load and the appearance of curvature of the periosteal and endocortical bones [30]. The basic function of the spinal column is to provide mechanical stability and protect the spinal cord. The vertebral body, as the bearer of the greatest load, together with the vertebral arch, form the spinal canal and protect the spinal cord from injuries. However, the diameter of the spinal canal is not uniform along its entire length and in humans its width increases from the fi rst to the last lumbar vertebra, while the depth is lowest at the L3 level and increases in a caudal direction [31]. Thus, in humans, there is a correlation between the diameter of the spinal canal and the place of greatest load on the spine, which can be considered a remarkable protective mechanism that aims to prevent pressure on the spinal cord that could occur when a higher mechanical force is applied. The results of our study show that in guinea pigs, the greatest depth of the spinal canal was measured at the level of L4, and the smallest in L6. CONCLUSION The results of our study indicate that in guinea pigs there is no linear increase in the values of morphometric parameters of the lumbar vertebrae in the direction from the fi rst to the last, as is the case in humans. In guinea pigs, L4 and L5 were observed to have the greatest body lengths, while the ventral side of the body of L4 was the most concave. Additionally, the depth of the spinal canal was greatest at the level of L4. This atypical morphology of the lumbar vertebrae leads to the conclusion that the direction of force transmission through the spinal column of tetrapedal organisms differs from that in bipedal organisms, and that the center of gravity of the load is shifted in a cranial direction. Also, the atypical morphology of L4 could indicate that this vertebra has the role of “shock absorber” and that it suffers the greatest load. However, it must be remembered that this conclusion was made on the basis of vertebral body morphometry alone, while the biomechanics of the spine are infl uenced by numerous other factors such as bone structure (direction of provision trabeculae and density), the characteristics of facet joints, intervertebral discs and associated soft tissue structures. Acknowledgments This work was predominantly funded by authors and to a lesser extent by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. 451-03-68/2022-14/200007). 67 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 Author’s contributions MM, SK, and MLM conceived of the study, participated in its design and coordinated the work. Morphometric measurements were performed by MM, SK, and AT. NT, MM, and SK performed the statistical analysis. MM, SK, and AT wrote the manuscript in consultation with MLM and NT. All authors read and approved the fi nal manuscript. 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Quant Imag Med Surg, 2016, 6(6): 671-679. 69 Acta Veterinaria-Beograd 2023, 73 (1), 55-70 RADIOLOŠKA ISPITIVANJA MORFOLOGIJE LUMBALNIH PRŠLJENOVA ZAMORACA (CAVIA PORCELLUS) – BIOMEHANIČKI ASPEKT Marko Jumake MITROVIĆ, Sara KITANOVIĆ, Nikola TATALOVIĆ, Anastasija TODOROVIĆ, Mirjana LAZAREVIĆ MACANOVIĆ Brojne studije su zasnovane na upotrebi animalnih modela, ipak, kod bipedalnih i te- trapedalnih organizama postoje značajne razlike u biomehanici kičmenog stuba, koje mogu značajno narušiti kvalitet i primenjivost dobijenih rezultata. Cilj ovog ispitivanja je da se dobiju osnovni podaci o morfometrijskim parametrima lumbalnih pršljenova zamoraca, čijom analizom će se ukazati na mesto najvećeg mehaničkog opterećenja. Morfometrijsko ispitivanje lumbalnih pršljenova zamoraca je sprovedeno na rendgen- skim snimcima 12 jedinki, sa jednakim brojem mužjaka i ženki. Rezultati ispitivanja pokazuju da lumbalni pršljenovi zamoraca imaju nepravilnu trapezoidnu geometriju i da su izmerene dužine L4 i L5 najveće. Parametri visine određivani su u medijalnom nivou i pokazali su da L4 ima najkonkavnije telo. Štaviše, L4 je imao najveću dubinu kičmenog kanala na istom nivou merenja. Shodno tome, kod zamoraca je najveće op- terećenje u L4 regiji za razliku od ljudi, gde se usled aksijalnog opterećenja kičmenog stuba najveći pritisak ostvaruje na poslednjim lumbalnim pršljenovima.

Journal

Acta veterinariade Gruyter

Published: Mar 1, 2023

Keywords: biomechanics; guinea pig; lumbar spine; morphometry; radiology

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