Breast tumor characteristics of BRCA1 and BRCA2 gene mutation carriers on MRI

The appearance of malignant lesions in BRCA1 and BRCA2 mutation carriers (BRCA-MCs) on mammography and magnetic resonance imaging (MRI) was evaluated. Thus, 29 BRCA-MCs with breast cancer were retrospectively evaluated and the results compared with an age, tumor size and tumor type matched control group of 29 sporadic breast cancer cases. Detection rates on both modalities were evaluated. Tumors were analyzed on morphology, density (mammography), enhancement pattern and kinetics (MRI). Overall detection was significantly better with MRI than with mammography (55/58 vs 44/57, P = 0.021). On mammography, lesions in the BRCA-MC group were significantly more described as rounded (12//19 vs 3/13, P = 0.036) and with sharp margins (9/19 vs 1/13, P = 0.024). On MRI lesions in the BRCA-MC group were significantly more described as rounded (16/27 vs 7/28, P = 0.010), with sharp margins (20/27 vs 7/28, P < 0.001) and with rim enhancement (7/27 vs 1/28, P = 0.025). No significant difference was found for enhancement kinetics (P = 0.667). Malignant lesions in BRCA-MC frequently have morphological characteristics commonly seen in benign lesions, like a rounded shape or sharp margins. This applies for both mammography and MRI. However the possibility of MRI to evaluate the enhancement pattern and kinetics enables the detection of characteristics suggestive for a malignancy.
Introduction
BRCA1 and BRCA2 are the most well known gene mutations responsible for an increased risk for developing breast cancer. A BRCA1 or BRCA2 mutation carrier (BRCA-MC) has approximately a 3% risk of getting breast cancer before the age of 30. This risk increases to almost 50% when she reaches the age of 50 and becomes 50–80% at the age of 70 [1, 2]. To reduce this risk, these women can choose between bilateral prophylactic mastectomy [3], oophorectomy [4] or chemoprevention [5]. In breast cancer, close surveillance contributes to a more favorable stage of disease at detection and may reduce the rate of death from breast cancer [6, 7].
In the surveillance or general screening for breast cancer, mammography still plays a prominent role. However, due to the young age and thus in most cases dense breast tissue, the sensitivity for mammography is moderate. False-negative rates of up to 62% have been reported for mammography in screening gene mutation carriers [8, 9]. A malignant lesion in the breast is mammographically best detected if it presents itself as an ill-defined or spiculated mass, a group of microcalcifications or as an architectural distortion. A smoothly outlined well-defined mass detected on mammography has a chance of less than 1% of being malignant [10, 11]. Tilanus et al. [12] and Kaas et al. [13, 14] have evaluated the mammographic appearance of breast cancer in BRCA-MC. Tilanus and coworkers found the mammographic appearance suspicious for a malignancy in only 38% of the gene carriers in comparison with 71% in a control group. “Prominent pushing margins” caused by a continuous front of tumor cells not separated by connective tissue were described in the BRCA-MC group as the main reason for a false-negative evaluation of mammograms [12]. Kaas and coworkers concluded in their study of 31 breast cancer cases in BRCA-MCs that all mammographically detected lesions should be further evaluated by ultrasound and biopsy regardless of their appearance [13]. Well-defined mammographic tumors correlated in 83% with histologic circumscribed tumor margins in BRCA1-MCs [14].
For women with an increased risk for developing breast cancer magnetic resonance imaging (MRI) should be included for close surveillance [15, 16]. The superior sensitivity of MRI (81%) for the detection of breast cancer in these women compared with mammography (40%) has been proven in literature [17]. However the classification of a lesion detected on MR as benign or malignant still remains a challenge. Morphological and dynamic features are important in breast MRI interpretation. Focal masses with smooth borders are associated with a high negative predictive value for malignancy [17]. An irregular lesion contour, inhomogeneous enhancement pattern and rim enhancement have been reported as features indicating malignancy [18]. The dynamic evaluation is often based on the enhancement characteristics 2–7 min after the injection of a paramagnetic contrast agent. In this approach, the decrease of signal intensity, often referred to as a type 3 curve or washout, is highly predictive for breast cancer, with a likelihood of malignancy of 87% [19]. Until now the appearance of breast malignancies in BRCA-MCs has only been investigated for mammography. In this study we analyzed the MRI characteristics of BRCA-MC-associated tumors compared with sporadic cases of breast cancer.
Materials and methods
All available (35) BRCA-MCs with a biopsy-proven malignancy, imaged with MRI for screening [9] or pre-operative evaluation in the period from July 2000 until November 2006, were included in the study: 23 BRCA1 carriers and 12 BRCA2 carriers. In order to compare tumor characteristics with sporadic cases of breast cancer an age, tumor type and tumor size matched control group was composed from 206 consecutive sporadic breast cancer cases imaged with MRI in the period from November 2001 until January 2007. All BRCA-MC cases were age matched within 5 years with sporadic breast cancer cases. Cases were also matched for tumor type (IDC, ILC or DCIS) and pathological tumor size. For size matching, the BRCA-MC cases were matched to the closest tumor size in the sporadic cases available, with a limit for the maximum size difference of 0.5 cm for tumors smaller than 1 cm, 1-cm difference for tumors up to 5 cm and 1.5-cm difference for tumors larger than 5 cm. BRCA-MC cases that could not be matched following these criteria were excluded.
Mammograms were obtained in the mediolateral oblique and craniocaudal direction on a digital mammographic unit (Senograph 2000 D or a Senograph DS, GE Healthcare, Wis., USA). Detection, density of the lesion compared with breast tissue, lesion morphology, and size were scored. In the morphologic assessment, lesion type was classified as either a mass, a calcifications or as an architectural distortion. Lesion shape was described as rounded, lobulated or irregular and lesion margins as sharp, vague or spiculated. The size of the tumor was measured by determining the longest axis through the displayed lesion. Spiculae surrounding a solid lesion were interpreted as desmoplastic reaction and not included in the measurement.
MRI investigations were performed on a 1.5-Tesla system with a double breast coil (Magnetom Vision, Sonata or Symphony, Siemens, Erlangen, Germany). In the scanning, we used a coronally orientated three-dimensional fast low-angle shot (FLASH 3D) with the following parameters: TE 4 ms, TR 8.1 ms, FA 20°, FOV 360 mm, TA 96 s, image resolution 1.5 mm × 1.5 mm × 1.5 mm for all patients scanned prior to June 2004 and TE 4 ms, TR 7.5 ms, FA 8°, FOV 320 mm, TA 87 s, image resolution 1.3 mm × 1.3 mm × 1.3 mm for all patients scanned after June 2004.
Prior to the MR examination, an intravenous catheter was inserted. All patients were placed in the prone position, with the breasts in the double breast coil and positioned at the isocenter of the magnet. After localizer images were obtained in three directions and a precontrast FLASH 3D series was recorded, 0.1 mmol/kg bodyweight gadolinium chelate (Magnevist, Schering, Germany or Dotarem, Guerbet, The Netherlands) was administered using a power injector (Spectris, Medrad, USA) at 2.5 ml/s followed by a 15-ml saline flush at the same injection rate. Thereafter, five post contrast FLASH 3D series were recorded.
All MRI examinations were retrospectively evaluated on a dedicated breast MRI workstation (Dynacad, Invivo, USA) scoring lesion detection, size, morphology and enhancement kinetics. Maximum intensity projections, coronal images and axial reconstructions of both the T1 weighted and subtracted images and time-intensity curves were displayed. The morphologic assessment included lesion shape, margin appearance and enhancement pattern. Lesion shape was classified as being rounded, lobulated or irregular. Margins were described as sharp, vague or spiculated. The enhancement pattern of a lesion was classified as homogeneous, heterogeneous or rim enhanced. Lesion enhancement kinetics were evaluated according to the criteria described by Kuhl et al. [19]. Type 1 shows persistent enhancement and is highly suggestive for a benign lesion. Type 2 shows a plateau after initial increased enhancement, where the maximum signal intensity is reached approximately 2–3 min after contrast injection and remains constant. This type of curve is seen in both benign and malignant lesions. In a type 3 curve, the peak enhancement is reached in the early postcontrast phase, and this is followed by a decrease of signal intensity (wash-out). The latter curve is strongly suggestive for a malignant lesion. The dynamic curves were evaluated based on a single voxel or by selecting a region of interest within the lesion, the workstation allowed the readers to use both methods. Because of the possible bias in this retrospective study, a BI-RADS classification [20] could not be scored objectively and was therefore not included in the evaluation.
All studies were evaluated retrospectively by two radiologists in conference and consensus. BRCA-MCs and controls were mixed during the evaluation. Except from the knowledge of a malignancy being present, the radiologists were blinded to any other clinical information. Mammography and MRI images were evaluated in separate sessions. From the histopathology reports, the tumor type, size and mitotic activity index (MAI) were recorded. The study was approved by the institutional review board; since the study was performed retrospectively, informed consent was not required according to the review board.
In the statistical evaluation, differences in patient and tumor characteristics between the BRCA-MC and control group were analyzed using an independent sample t-test if variables were continuous and normally distributed. For categorical variables, the Pearson chi-square test was used and we used Fisher’s exact test when any of the expected values was less than five. Pearson’s correlation coefficients were calculated for both mammographic size and MRI size versus pathologic size. All statistical analyses were performed using SPSS statistical software (version 12.0.1). P values <0.05 were considered to indicate statistical significance.
Results
Six BRCA-MC cases could not be matched according to the criteria defined; these cases were excluded from the study. Four BRCA-MCs were excluded because no match could be found based on patient’s age; the other two were excluded because no match could be found based on tumor size.
The mean age and tumor size of the excluded cases were, respectively, 33 years (range 27–36, median 35, SD 3.4 years) and 1.4 cm (range 0.6–2.8 cm, median 1.1 cm, SD 0.8 cm). A total of 29 BRCA-MC cases were included for this study. In the BRCA-MC group, five women were symptomatic (17%); 21 women were symptomatic in the control group (83%).
Mean age in the BRCA-MC group was 42 years (range 32–68 years, median 40 years, SD 8.0 years); this was 44 (range 37–64 years, median 43 years, SD 5.6 years) for the control group. The mean pathological tumor size was 2.0 cm (range 0.4–7.0 cm, median 1.4 cm, SD 1.5 cm) in the BRCA-MC group and 2.3 cm (range 0.6–7.0 cm, median 1.9 cm, SD 1.7 cm) in the control group. No significant difference was found for patient age (P = 0.289) or maximal pathological tumor size (P = 0.371).
The mean tumor size on mammography was 2.1 cm (range 0.5–7.0 cm, median 1.5 cm, SD 1.49 cm). The mean tumor size on MRI was 2.4 cm (range 0.6–7.1 cm, median 1.8 cm, SD 1.75 cm).
There was a significant correlation between imaging measurements and pathological measurements; 0.664 (P < 0.001) for mammographic measurements and 0.808 (P < 0.001) for MRI measurements.
In both the BRCA-MC and control groups, 23 cases were based on invasive ductal carcinoma (IDC), two cases on invasive lobular carcinoma (ILC), one case on ductal carcinoma in situ grade 1 (DCIS1) and three cases on ductal carcinoma in situ grade 2 (DCIS2). In the BRCA-MC group, a mean MAI of 33.0 (range 6–100, median 27, SD 27.1) was found, compared with 17.5 (range 1–60, median 14, SD 15.9) in the control group. The difference in MAI between the BRCA-MC and control groups was found to be significant (P = 0.044).
In the control group, one patient refused to undergo mammography because of implants. Overall mammography detected 44 of 57 lesions and MRI detected 55 of 58 lesions. Therefore, the overall detection is significantly better with MRI than with mammography (P = 0.021).
Mammography detected 22 (76%) lesions in the BRCA-MC group and 22 (79%) in the control group. No significant difference was found (P = 0.807). All lesions missed on mammography in the BRCA-MC were IDC. In the control group, five cases of IDC and one case of ILC were missed. Mammographic lesion characteristics are presented in Table 1. Lesions in the BRCA-MC group were significantly more described as rounded (12//19 vs 3/13, P = 0.036) and were more often described to have sharp margins (9/19 vs 1/13, P = 0.024). Lesions in the control group were significantly more described as irregular (10/13 vs 6/19, P = 0.029). From the six BRCA-MCs that were excluded, only three lesions were detected on mammography. In two cases a mass was detected and in one case calcifications. Both these masses were described as rounded with sharp margins.
Table 1Mammographic lesion characteristics for both groups BRCA-MC groupControl groupP valueLesions detectedn = 22n = 22Lesion typeMass19130.042Arch. distortion140.345Calcification250.216Lesion densityaHyperdense810.050Isodense11120.050Lesion morphologyaRounded1230.036Lobulated1–1.000Irregular6100.029Lesion marginsaSharp910.024Vague880.473Spiculated240.194aLesion density, morphology and margins of mass-like lesions only
On MRI, 27 lesions (93%) were detected in the BRCA-MC group, 28 (97%) in the control group. No significant difference was found (P = 0.553). The lesions missed in the BRCA-MC group were both cases of DCIS, one case of DCIS1, seen on mammography as an architectural distortion, and one case on DCIS2, seen on mammography as a mass. The lesion missed in the control group was based on DCIS2, seen on mammography as calcifications. Their was no significant difference found for the detection of breast cancer between mammography and MRI within the BRCA-MC (P = 0.18) or the control group (P = 0.13). Morphological and dynamic MR characteristics are presented in Table 2. Lesions in the BRCA-MC group were significantly more often described as rounded (16/27 vs 7/28, P = 0.010), with sharp margins (20/27 vs 7/28, P < 0.001) and to show rim enhancement (7/27 vs 1/28, P = 0.025). Lesions in the control group were significantly more often described as irregular (18/28 vs 8/27, P = 0.010), with vague margins (15/28 vs 6/27, P = 0.017) and with a heterogeneous enhancement pattern (22/28 vs 12/27, P = 0.009). No significant difference between the two groups was found for enhancement kinetics (P = 0.667). From the six BRCA-MCs that were excluded, five were detected on MRI. Four of these lesions were described as rounded, one as irregular. The delineation was described as sharp in four and as vague in one of these cases. The enhancement pattern was described as homogeneous in two, heterogeneous in one and as rim-enhancement in two of these cases. All five cases showed a type 3 curve.
Table 2MRI lesion characteristics for both groups BRCA-MC groupControl groupP valueLesions detectedn = 27n = 28Lesion morphologyRounded1670.010Lobulated331.000Irregular8180.010Lesion marginsSharp207<0.001Vague6150.017Spiculated160.049Enhancement patternHomogeneous850.304Heterogeneous12220.009Rim710.025Enhancement kineticsType 1121.000Type 2460.525Type 322200.380
Discussion
In this study, the overall false-negative rate for mammography was significantly higher compared with MRI. Although it is expected that the sensitivity for MRI in both the control group and the BRCA-MC is higher compared with mammography, no significant difference was found in this study within the two groups. This is probably due to the relatively small numbers of cases in this study. Kaas et al. [13] described in their mammographic study on BRCA1 and BRCA2 mutation carriers a sensitivity of 64% for the detection of a tumor in the original reports. In this study, 76% of the lesions were visible on mammography. Since this was a retrospective study and the radiologist was aware of the fact that a tumor was present at the time of the evaluation, no conclusion can be drawn from the difference in detection between both studies.
On mammography, the mass like lesions detected in the BRCA-MC group were significantly more often described as rounded. Also, lesions were found to differ in margin appearance; tumor margins in the BRCA-MC group were significantly more often described as sharp. A smooth, nonspiculated mass has previously been described by Tilanus et al. [12] as a reason for a false-negative mammographic evaluation in BRCA-MC. Thus, although Sickles et al. [21] described that nonpalpable, circumscribed, noncalcified breast masses (probably benign) should be managed with periodic mammographic surveillance regardless of lesion size and patient age, and Sardanelli et al. [22] published that both the well-defined margins and the rounded shape are more often associated with benign lesions, these findings are not applicable to the BRCA-MCs studied in this or other studies on this subject. Tilanus et al. [12] stated that any mammographic mass in BRCA-MCs must be regarded with suspicion. A similar conclusion was published by Kaas et al. [13]. An additional evaluation using ultrasound and biopsy of all lesions detected in BRCA-MC is mandatory, regardless of their morphological appearance. In their study of 28 BRCA-MCs, Hamilton et al. [23] also described the appearance of breast tumors on ultrasound. On ultrasound, 53% of the tumors were classified as either benign or indeterminate, making a biopsy of any detected mass inevitable. We are also of the opinion that any solid lesion detected in BRCA-MCs should be evaluated by a biopsy.
In the screening of women with an increased risk for developing breast cancer, more tumors are detected by MRI compared with mammography [8, 9]. In this study, all lesions missed on MRI were cases of DCIS. MRI is known to have a lower sensitivity in detecting DCIS compared with invasive carcinomas [24], especially in low-grade DCIS. The low or intermediate contrast uptake that is often observed in pure DCIS and the absence of a type 3 curve can result in a false-negative evaluation [24]. Kriege et al. [9] found in the screening of 1,909 women with an increased risk for developing breast cancer, including 358 carriers of germ-line mutations, that MRI missed five cases of DCIS that were detected on mammography, with six noninvasive tumors detected in total in the study. However, Kuhl et al. [25] reported MRI to be more sensitive for DCIS compared with mammography in a prospective study of women with an increased risk for developing breast cancer. Although MRI proved to be more sensitive for the detection of DCIS compared with mammography, not all cases of DCIS were detected by either modality. Therefore, at this point both modalities are still needed in the screening of women with an increased risk for developing breast cancer.
Similar to mammography, on MRI a significantly higher number of lesions were described as rounded and with sharp margins in the BRCA-MC group. Furthermore, the number of lesions with ‘rim-enhancement’ was found to be significantly higher in the BRCA-MC group. The presence of this enhancement pattern has been associated with malignant lesions [26, 27]. Because MRI enables the radiologist to evaluate the enhancement pattern of the lesion, where mammography does not, these lesions will become more suspectedly malignant, even though other morphologic features are more often seen in benign lesions. The association of rim-enhancement (Fig. 1) with central necrosis or insufficient microvessel growth can be an indicator for the growth rate of tumors. Jimenez and coworkers have described centrally necrotizing carcinomas to have an accelerated clinical course and early systemic metastasis [28]. An accelerated growth rate can be associated with a high MAI. In this study, the MAI was found to be significantly different between both groups. This is in agreement with Tilanus et al. [12], who also found the mitotic count to be significantly higher in tumors found in gene mutation carriers. The higher rate of sharp tumor margins and rim-enhancement may thus be explained by the more aggressive nature of tumors in BRCA-MCs. Several authors have implied that, due to the rapid growth rate of tumors in gene mutation carriers, the screening frequency should be adjusted [13, 29]. Komenaka et al. [29] suggest a higher screening frequency in carriers because of the high number of interval cancers found in their group. Half of these interval malignancies were already positive for lymph-node involvement. The 13 carriers in their study were screened with mammography. Mammography in carriers is not sensitive, particularly because the women are young and thus more often have dense breasts [30, 31]. The use of MRI in screening has already been a step forward since MRI can detect smaller tumors, often occult for mammography, that are less likely to have progressed into lymph-node involvement [9].
Fig. 1a An MLO mammogram from the right breast of a 42-year-old BRCA1-mutation carrier. b A coronal subtraction MR image of the same breast. An 11-mm sharply delineated rounded lesion is present on the mammogram projecting over the upper quadrants (arrow). On MRI, the same lesion was detected (arrow) with rim-enhancement. The rim-enhancement makes this lesion morphologically suspect malignant. Ultrasound guided core biopsy proved this lesion to be an invasive duct carcinoma
Rounded, homogeneous enhancing lesions found on MRI are in general not considered suspicious. A homogeneous enhancement pattern, found in nine BRCA-MCs, does not contribute to the malignant nature of these lesions. Therefore, enhancement kinetics are of value. In this study, the dynamic analysis showed in both the BRCA-MC and the control groups a type 3 curve in, respectively, 82% and 71% of the cases. As described by Kuhl et al. [19], a type 3 enhancement curve is highly indicative for malignancy. Using this characteristic, even rounded, sharply delineated, homogeneous enhancing lesions become suspect malignant (Fig. 2).
Fig. 2a A coronal subtraction MR image from the right breast of a 49-year-old BRCA1-mutation carrier. Lateral located in the right breast, a sharply delineated rounded, homogeneous enhancing lesion is visible with a longest diameter of 9 mm (arrow). b The relative enhancement versus time curve. The type 3 curve seen for this lesion was the only characteristic indicating a possible malignancy. Pathology proved this lesion to be an invasive duct carcinoma
Despite all the findings discussed in this study, it remains questionable if the characterization of a lesion detected on either mammography or MRI in BRCA-MCs is even necessary. As the chance for these women to develop breast cancer is significantly increased, almost any detected lesion will in practice be classified as suspect malignant until proven otherwise. Sardanelli et al. [17] found a positive predictive value for MR in women with an increased risk of only 53% due to a high number of false positives. As stated previously for mammographically detected lesions, additional evaluation by core biopsy is the only definitive classification for lesions detected in this group of women. In the case of MRI screening in high risk women, short-term follow-up, target ultrasound or MRI-guided biopsies are therefore often indicated [32]. What the best strategy in this group of women will be, also in terms of cost effectiveness, needs to be further studied.
We conclude that in BRCA-MC malignant lesions frequently have morphological characteristics that are commonly seen in benign lesions, like a rounded morphology or a sharp delineation. This applies for both mammography and MRI. However, the possibility of MRI to evaluate the enhancement pattern and enhancement kinetics of lesions enables the radiologist to detect characteristics suggestive for a malignancy.


Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination

Background Vascular calcification is an organized process in which vascular smooth muscle cells (VSMCs) are implicated primarily. The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro. Methods VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol/l β-glycerophosphate, 8 mmol/l CaCl2, 10 mmol/l sodium pyruvate, 1 μmol/l insulin, 50 μg/ml ascorbic acid, and 100 nmol/l dexamethasone (calcification medium). Calcification, proliferation, and apoptosis of VSMCs were quantified. Results Calcium deposition was stimulated dose-dependently by β-glycerophosphate, CaCl2, and ascorbic acid (all P < 0.01). Addition of amlodipine (0.01–1 μmol/l) to the calcification medium did not affect VSMC calcification. However, atorvastatin (2–50 μmol/l) stimulated calcium deposition dose-dependently. Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone. Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used. Only atorvastatin (50 μmol/l) induced considerable apoptosis of VSMCs. Conclusion In vitro calcification of VSMCs is not affected by amlodipine, but is stimulated by atorvastatin at concentrations ≥10 μmol/l, which could contribute to the plaque-stabilizing effect reported for statins.
Introduction
Atherosclerosis is the principal cause of coronary artery disease, stroke, and peripheral artery disease, and is the major cause of mortality in the Western hemisphere. Vascular calcification is a prominent feature of atherosclerosis, and it is associated with an increased risk of myocardial infarction [1]. Vascular calcification refers to the deposition of calcium phosphate mineral, most often in the form of hydroxyapatite, in the vessel wall. Calcification of the vessel wall and heart valves is associated with ageing, diabetes, and uremia [2–4].
Vascular calcification is now considered to be an organized, regulated process comparable to bone mineralization. The presence of various components associated with bone mineralization, such as bone morphogenetic protein, osteocalcin, osteopontin, osteoblast-like cells, and matrix vesicles in atherosclerotic lesions supports this concept [5–7]. Vascular cells such as vascular smooth muscle cells (VSMCs) and pericyte-like cells play an important role in vascular calcification [8].
The lipophilic calcium antagonist (CA) amlodipine has been shown to restore cholesterol-induced membrane bilayer abnormalities in VSMCs derived from the atherosclerotic rabbit aorta [9, 10], thereby restoring normal calcium homeostasis. Other mechanisms, through which CAs may affect atherosclerosis development include inhibition of proliferation and migration of VSMCs [11, 12], and inhibition of lipoprotein oxidation [13]. In addition, CAs modify binding of monocytes to the endothelium, and activate synthesis of matrix components [14]. The effects of CAs on atherosclerotic calcification have not been widely studied, however.
Lipids may contribute to atherosclerotic calcification [15, 16]. Recently statins have been shown to decrease the progression of coronary artery calcification and aortic valve calcification [17, 18]. Therapy with a combination of a CA and a statin might be more atheroprotective than either treatment alone [19, 20]. Combination therapy with CA and statin was shown to improve endothelial function and arterial compliance, to diminish LDL atherogenicity, and to slow the progression of atherosclerosis. So far, the effects of the combination therapy with CA and statin on vascular calcification have not been studied extensively.
We used an in vitro model of vascular calcification by neonatal rat VSMCs, isolated by outgrowth from aortic explants. The purpose of this investigation was to identify the factors, and their concentrations, that are mandatory to induce calcification of aortic VSMCs in vitro. Since calcium is such a major component of advanced atherosclerotic lesions, we furthermore studied whether the CA amlodipine and the statin atorvastatin, alone and in combination, affected calcification of aortic VSMCs in vitro.
Materials and methods
Cell culture
Vascular smooth muscle cells (VSMCs) were obtained from segments of aortas explanted from 2-day old Wistar rats. Aortic segments were obtained aseptically and cut open longitudinally. The endothelium was removed by gently rubbing the luminal side of the aortas over the surface of a tissue culture dish (Falcon). Subsequently, the aortas were placed, lumen side down, on the bottom of a tissue culture flask (Greiner), and allowed to adhere for approximately 3 h. Then tissues were immersed in growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies), penicillin (100 U/ml), and streptomycin (100 μg/ml) (both supplied by BioWhittaker Europe). Seven days later the aortic tissues were removed, and the VSMCs that had grown out were detached by trypsinization. The detached cells were resuspended in growth medium and seeded in tissue culture flasks (Greiner), 6 or 12-well plates or on glass cover slips.
Immunocytochemistry
Subconfluent cultures at early passage were examined for the presence of α-smooth muscle actin, SM myosin heavy chain and calponin using specific antibodies (anti-SM actin clone 1A4 (1:400), Sigma; anti-SM myosin heavy chain (1:100), SanverTech; anti-calponin (1:10,000), Sigma). After fixation in 1% formalin in PBS on ice for 30 min, cells were permeabilized with 0.1% Triton X-100 for 30 min, and subsequently incubated with the primary antibody for several hours at 4°C. Secondary antibodies were conjugated with fluorescein isothiocyanate (FITC) or Cy3 (Sigma). To identify cell nuclei, the cells were also stained with Hoechst 33342 (10 μg/ml; Molecular Probes). Immunofluorescent images were obtained using a fluorescence microscope (Nikon Eclipse) equipped with 20×, 40×, and 100× objectives and a digital camera (Nikon DXM1200).
In vitro calcification of VSMCs
Calcification of VSMC cultures was induced by the method of Shioi et al. [21] with minor modifications. When confluent, the incubation medium of VSMCs was switched from growth medium to calcification medium. Calcification medium consisted of DMEM (high glucose, 4.5 g/l) supplemented with 15% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), 8 mmol/l CaCl2, 10 mmol/l sodium pyruvate, 1 μmol/l insulin, 50 μg/ml ascorbic acid, 10 mmol/l β-glycerophosphate, and 100 nmol/l dexamethasone. The medium was replaced with fresh medium every 2–3 days. In several experiments, the concentrations of CaCl2, ascorbic acid, β-glycerophosphate, and dexamethasone were varied.
In another series of experiments, calcification medium was supplemented with amlodipine (0.01–1 μmol/l), atorvastatin (2–50 μmol/l), or a combination of these drugs. The CA amlodipine was dissolved in absolute ethanol at a concentration of 1 mmol/l. This stock solution was to be diluted at least 1:1,000 in culture medium. Atorvastatin, an inhibitor of HMG-CoA reductase, was dissolved in absolute ethanol at a concentration of 10 mmol/l. This stock solution was to be diluted at least 1:200 in culture medium.
Cells treated with calcification medium without added drugs were used as controls. Appropriate amounts of solvent (ethanol) were added to these controls. Addition of ethanol at these levels did not affect cell growth. After 3 weeks of incubation, calcification was quantified.
Assessment of calcium deposition
VSMCs were decalcified with 0.6 N HCl for 24 h. The calcium contents of the supernatants were determined by spectrophotometer using the o-cresolphthalein method (Roche Diagnostics). After decalcification, the cells were washed with PBS and scraped from the culture plate. The protein content was measured using the BCA protein assay (Pierce). The calcium content of the cell layer was normalized to protein content.
Proliferation assay
VSMCs were seeded at a density of 5 × 103–2 × 104 cells/well in a 96-well plate and allowed to attach overnight. Subsequently, the standard culture medium (DMEM, 10% FBS, antibiotics) was replaced by calcification medium supplemented with amlodipine, atorvastatin, or a combination of both. Cell proliferation was assessed at day 4 and day 9 using the Cell Proliferation Kit II (XTT, Roche), which is a colorimetric assay for the non-radioactive quantification of cell proliferation and viability.
Detection of apoptosis
To identify apoptotic cells by assessment of condensation of nuclear chromatin, VSMC cultures were stained with Hoechst 33342 (10 μg/ml; Molecular Probes) for 10 min in the dark. Immunofluorescent images were obtained using a fluorescence microscope (Nikon Eclipse) equipped with 20x, 40x, and 100x objectives and a digital camera (Nikon DXM1200). In 6 frames per treatment, an average of 250 nuclei per frame were assessed for the presence of apoptosis.
Statistical analysis
Results are expressed as mean ± SEM. For statistical analysis, SPSS 10.0 for Windows was used. Since the data were not normally distributed, non-parametric tests were used for comparisons between groups. Overall comparisons between groups were performed with the Kruskall–Wallis test. If only two groups were compared, Mann–Whitney rank sum tests were used. P values less than 0.05 were regarded as significant.
Results
VSMCs that have been cultured from rat aortic explants for approximately 7 days stained positive for α-smooth muscle-actin, smooth muscle myosin heavy chain, and calponin, confirming the smooth muscle cell nature of these cells (Fig. 1, online supplement). VSMCs of passage 3–6 were subsequently used in calcification experiments.
Fig. 1(A) Rat aortic VSMC isolated by outgrowth, phase contrast. Immunofluorescent staining of neonatal rat VSMC incubated with (B) (and insert) anti-smooth muscle actin Ab, (C) anti-smooth muscle myosin Ab, and (D) anti-calponin Ab. Original magnification B and C ×100, D and insert ×200. VSMC = vascular smooth muscle cell
Calcium deposition and dependence of extracellular Ca2+ concentration
Cells were incubated with calcification medium supplemented with various Ca2+ concentrations for 21 days. At Ca2+ concentrations ≤3 mmol/l, hardly any calcium deposition was observed, but at Ca2+ concentrations >3 mmol/l, a dose-dependent increase in calcium deposition was observed (Fig. 2A). The amount of calcium deposition was positively correlated to calcium concentration in the culture medium (P < 0.01). We chose to continue with 8 mmol/l of Ca2+-ions added to the calcification medium.
Fig. 2(A) Dose-dependent effects of CaCl2 on calcification of neonatal rat VSMCs. (B) Dose-dependent effects of β-glycerophosphate on calcification of neonatal rat VSMCs. VSMCs were treated for 21 days with calcification medium containing varying concentrations of Ca2+ ions or β-glycerophosphate. Control cultures (=con) were incubated with DMEM, 10% FBS, and antibiotics. Calcium deposition was quantified by o-cresolphthalein method. The data are presented as mean ± SEM (n = 3–9). *P < 0.05 vs. control cultures. ◆P < 0.05 vs. 2 and 3 mM calcium in culture medium. #P < 0.05 vs. all other treatments
Calcium deposition and dependence of extracellular phosphate concentration
Shioi et al. have demonstrated that β-glycerophosphate accelerates in vitro calcification of VSMCs and induces extensive calcium deposition in a manner analogous to in vitro mineralization by osteoblasts [21]. In our model of vascular calcification, the amount of calcium deposition was positively correlated to the β-glycerophosphate concentration in the calcification medium (P < 0.01; Fig. 2B).
Calcium deposition and dependence of extracellular dexamethasone concentration
To determine the contribution of added dexamethasone, we incubated the VSMCs with various concentrations of dexamethasone (10–1,000 nmol/l). No significant differences in calcium deposition were observed between the cells incubated with 0, 10, 100, and 1,000 nmol/l dexamethasone (Fig. 3A).
Fig. 3(A) Dose-dependent effects of dexamethasone on calcification of neonatal rat VSMCs. (B) Dose-dependent effects of ascorbic acid on calcification of neonatal rat VSMCs. VSMCs were treated for 21 days with calcification medium containing varying amounts of dexamethasone or ascorbic acid. Calcium deposition was quantified by o-cresolphthalein method. The data are presented as mean ± SEM (n = 6–12). #P < 0.05 vs. all other treatments. ◆P < 0.05 vs. 0 and 5 μg/ml ascorbic acid
Calcium deposition and dependence of extracellular ascorbic acid concentration
To determine the contribution of ascorbic acid to calcium deposition, we incubated VSMCs with various concentrations of ascorbic acid (0, 5, 50, and 500 μg/ml). At the highest concentration (500 μg/ml) ascorbic acid was associated with significantly more calcium deposition than at all other concentrations (Fig. 3B). However, 500 μg/ml ascorbic acid caused a considerable decrease in pH of the culture medium. Since calcium deposition was positively correlated with ascorbic acid concentration (P < 0.01), we decided to use 50 μg/ml ascorbic acid in future calcification studies, the highest concentration of ascorbic acid that did not cause acidification of the medium.
Effects of amlodipine on in vitro VSMC calcification
To study the effect of the CA amlodipine on VSMC calcification, VSMCs were incubated for 2–3 weeks with calcification medium supplemented with various concentrations of amlodipine (0.01–1 μmol/l). Incubation of neonatal rat VSMCs with amlodipine had no effect on VSMC calcification, at none of the concentrations tested (Fig. 4A).
Fig. 4 (A) Dose-dependent effects of amlodipine on calcification of neonatal rat VSMCs. (B) Dose-dependent effects of atorvastatin on calcification of neonatal rat VSMCs. (C) Effects of amlodipine, atorvastatin and a combination of both treatments on neonatal rat VSMC calcification. VSMCs were treated for 21 days with calcification medium containing varying concentrations of atorvastatin or amlodipine, a combination of both, or none of them (control). Calcium deposition was quantified by o-cresolphthalein method. The data are presented as mean ± SEM (n = 15). *P < 0.05 when compared to untreated control cultures. #P < 0.05 when compared to all other treatments
Effects of atorvastatin on in vitro VSMC calcification
To study the effect of the statin atorvastatin on VSMC calcification, VSMCs were incubated for 2–3 weeks with calcification medium supplemented with various concentrations of atorvastatin (2–50 μmol/l). Atorvastatin increased VSMC calcification dose-dependently (Fig. 4B). At a concentration of 2 μmol/l atorvastatin, calcium deposition was increased by 30% (P = 0.04) when compared to VSMCs incubated with atorvastatin-free calcification medium. At concentrations of 10 and 50 μmol/l atorvastatin, calcium deposition was increased 2.3-fold and 6.1-fold, respectively (P < 0.001) when compared to VSMCs incubated with atorvastatin-free calcification medium.
Effect of a combination of CA and statin on in vitro VSMC calcification
To study the effect of combining amlodipine and atorvastatin treatment on VSMC calcification, VSMCs were incubated for 2–3 weeks with calcification medium supplemented with 0.1 μmol/l amlodipine, 10 μmol/l atorvastatin, or a combination of these drugs in the same concentrations. Incubation of VSMCs with 0.1 μmol/l amlodipine had no effect on VSMC calcification. Incubation of VSMCs with 10 μmol/l atorvastatin resulted in a 2.2-fold increased calcium deposition when compared to control cultures treated with calcification medium only (Fig. 4C) (P < 0.001). Treatment with a combination of amlodipine and atorvastatin also resulted in a 2.2-fold increased calcium deposition when compared to control cultures treated with calcification medium only (P = 0.026). The combination therapy resulted in significantly more calcium deposition than treatment with amlodipine alone (P = 0.003), and as much calcium deposition compared to treatment with atorvastatin alone (n.s.).
Effects of amlodipine, atorvastatin, and their combination on VSMC proliferation
VSMC proliferation was assessed at day 4 and day 9. After 4 days of incubation in calcification medium supplemented with amlodipine (1 μmol/l), VSMC proliferation was decreased by 21% (P < 0.001) as compared to proliferation of VSMCs cultured in amlodipine-free calcification medium (Fig. 6A, online supplement). After 9 days of incubation, VSMC proliferation had increased by 14% (P < 0.05) when treated with 0.01 μmol/l amlodipine, but at an amlodipine concentration of 1 μmol/l, VSMC proliferation had decreased by 26% (P < 0.01) as compared to proliferation of VSMCs cultured in amlodipine-free calcification medium (Fig. 5A). Atorvastatin, at a concentration of 50 μmol/l, decreased VSMC proliferation by 50% (P < 0.001; Fig. 5B). Treatment of VSMCs with the combination of 0.1 μmol/l amlodipine and 10 μmol/l atorvastatin did not result in significant changes in cell proliferation compared to VSMCs incubated in the absence of these drugs.
Fig. 5Effect of amlodipine and atorvastatin on VSMC proliferation. VSMC proliferation was assessed at day 4 and day 9 using the Cell Proliferation Kit II (XTT)(Roche). VSMC proliferation is depicted as percentage relative to untreated cells (=100%) (A) Effect of amlodipine. (B) Effect of atorvastatin. #P < 0.05 vs. control cultures. *P < 0.05 vs. control cultures
Effects of amlodipine, atorvastatin, and their combination on VSMC apoptosis
VSMCs incubated in calcification medium only and VSMCs incubated with amlodipine (0.01–1 μmol/l) or atorvastatin (2 and 10 μmol/l) in calcification medium for 72 h hardly contained any apoptotic nuclei. Amlodipine at the highest concentration tested (1 μmol/l), caused apoptosis of on average 0.05% of the cells (n.s. compared to control). At the highest concentration of atorvastatin (50 μmol/l), however, 5.6% of the nuclei were apoptotic (P < 0.05 vs. control, 2 and 10 μmol/l atorvastatin; Fig. 6).
Fig. 6VSMCs were treated with 1 μmol/l amlodipine (A, B) or 50 μmol/l atorvastatin (C, D) for 72 h. Nuclei were visualized with Hoechst 33342. Apoptotic nuclei are circled in panel C and indicated with arrows in panel D. Microscopy: magnification A and C ×100, B and D ×400
VSMCs treated with the combination of 0.1 μmol/l amlodipine and 10 μmol/l atorvastatin for 72 h had a number of apoptotic nuclei that did not significantly differ from those observed in 10 μmol/l atorvastatin only, 0.1 μmol/l amlodipine only, and VSMCs incubated in calcification medium in the absence of these drugs.
Discussion
In an in vitro model of vascular calcification of rat aortic VSMCs, calcium deposition was dependent upon the extracellular concentration of organic phosphate (P < 0.01), calcium ions (P < 0.01), and ascorbic acid (P < 0.01). While studying the effect of the CA amlodipine and the statin atorvastatin, alone and in combination, on calcification, we found that, at none of the concentrations tested (0.01–1 μmol/l) did amlodipine have any effect on VSMC calcification in this model, nor on development of apoptosis. In contrast, atorvastatin stimulated VSMC calcification at a concentration of ≥10 μmol/l (P < 0.05), and 50 μmol/l inhibited VSMC proliferation and induced apoptosis (P < 0.05).
VSMCs retain remarkable plasticity, even in adult animals. VSMCs can undergo rapid and reversible changes in its phenotype in response to changes in local environmental conditions [22]. Due to production of a variety of cytokines by subendothelial macrophages, proliferation, and migration of VSMCs to the intimal layer can occur, thereby contributing to the process of intimal thickening and atherosclerosis.
Neonatal VSMCs resemble VSMCs in atherosclerotic plaque, since they retain the proliferative phenotype. Neonatal aortic explants exhibit rapid outgrowth of VSMCs, and we chose to use these cells to develop a model of vascular calcification. VSMCs were shown to migrate from the aortic explant within several days, and were identified as VSMCs by immunohistochemistry [8].
Dialysis patients have accelerated atherosclerosis, with extensive calcification of both the intima and the media [23]. Hyperphosphatemia has been implicated in this process [24]. Inorganic phosphate (Pi) levels have previously been shown to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro [25, 26]. In the present study, the amount of calcium deposition in neonatal rat VSMCs correlated positively to the β-glycerophosphate concentration in the calcification medium.
In the present study, the amount of calcium deposition in rat aortic VSMCs correlated to the calcium concentration in the calcification medium. This observation was not a result of passive calcium deposition, since incubation of VSMCs with normal growth medium supplemented with 8 mmol/l CaCl2 (final concentration) did not result in calcium deposition by these cells.
Shioi et al. [21] have previously demonstrated that culturing bovine VSMCs in the presence of β-glycerophosphate, ascorbic acid, and insulin can induce diffuse calcification in a manner analogous to in vitro mineralization of osteoblasts. The present study also showed that β-glycerophosphate is crucial for the induction of calcification in rat aortic VSMCs. Ascorbic acid augments this process. Dexamethasone was demonstrated to be less vital in this model of aortic VSMC calcification.
Statins possess potent lipid-lowering effects. Besides, statins exert pleiotropic effects on vascular wall cells, which include improvement of endothelial function, stabilization of the atherosclerotic plaque, and suppression of inflammation [27, 28]. Although several studies have failed to demonstrate an effect of statins on the progression of calcific aortic stenosis [29, 30], statins have also been shown to decrease the progression of coronary artery calcification and aortic valve calcification [17, 18, 31, 32]. Accordingly, Kizu et al. have demonstrated that statins inhibit calcification in an in vitro model of inflammatory vascular calcification [33]. Using interferon-γ, 1α, 25-dihydroxyvitamin D3, tumor necrosis factor-α, and oncostatin M to induce calcification in human VSMCs, it was demonstrated that cerivastatin and atorvastatin inhibited calcification dose-dependently. However, one should realize that this model differs from the model we have used in the present study. Whereas Kizu et al. used inflammatory mediators to induce calcification of the VSMCs, we used increased levels of CaCl2 and organic phosphate in the culture medium to promote calcification. Statins are known to have anti-inflammatory effects, and therefore, their inhibitory effect on calcification induced by inflammatory mediators is to be expected. In addition, low doses of statins have also been shown to protect human aortic VSMCs from inorganic phosphate-induced calcification [34].
In contrast to the aforementioned studies, the present study showed a dose-dependent stimulatory effect of atorvastatin (10 and 50 μmol/l) on calcification of VSMCs incubated in calcification medium. However, the dose of atorvastatin used in the present study was much higher than in the study described by Son et al. (500x higher at the highest dose) [34], and most likely explains the discrepancy between the results obtained by Son et al. and the results of the present study.
Reynolds et al. [35] have demonstrated that human VSMCs undergo vesicle-mediated calcification in response to changes in calcium and phosphate concentrations in the culture medium. Elevated calcium and phosphate concentrations resulted in increased release of vesicles and stimulation of apoptosis. Calcification was initiated by release of membrane-bound matrix vesicles from living cells and also by apoptotic bodies from dying cells. Vesicles released by VSMCs after prolonged exposure to calcium and phosphate contained preformed basic calcium phosphate, and calcified extensively. The present study confirms that statins stimulate apoptosis of VSMCs. Apoptotic bodies can calcify extensively [36], so a likely mechanism of induction of calcification by atorvastatin is through induction of apoptosis. Indeed, atorvastatin stimulated both apoptosis and calcification in our model.
Myocardial infarction and unstable angina usually arise from the disruption of mildly stenosed atherosclerotic lesions. Such vulnerable plaques typically contain a large amount of lipid and have a preponderance of inflammatory cells at the shoulders of the plaque and a thin fibrous cap. Calcium is found infrequently in the culprit lesion of ruptured plaque. Thus, plaque vulnerability is not associated with size, but with composition. Beckman et al. [37] presented the concept “that calcium is not a critical substrate for plaque disruption and is, in fact, associated with more stable plaques.” Therefore, statin-induced alterations in the plaque’s composition from lipid to fibrous tissue without or with calcification would decrease the plaque’s vulnerability to rupture, thereby decreasing the risk of acute coronary syndromes.
Prolonged intensive lipid-lowering has been demonstrated to increase calcium content of plaques as assessed by MRI in vivo, and lowered plaque lipid content at about the same extent, without a pronounced effect on plaque size [38]. Since atherosclerotic plaque composition plays an important role in plaque stability, with lipids destabilizing the plaque and calcification stabilizing the plaque, the effect of statins on plaque composition may be favorable. In addition to lipid-lowering and calcification-inducing effects, we observed that statins have an anti-proliferative effect on VSMCs. Less proliferation of VSMCs may destabilize the plaque. However, this only occurred at high doses of atorvastatin, whereas calcification induction occurred at lower dose of atorvastatin.
Although several in vitro and in vivo studies have demonstrated that combining CA and statin therapy might be more atheroprotective than either treatment alone [19, 20, 39], the present study found no difference between the combination of amlodipine and atorvastatin therapy, and atorvastatin therapy alone on VSMC calcification.
In conclusion, extracellular β-glycerophosphate and CaCl2 concentrations are important determinants of in vitro calcification of rat aortic VSMCs. Ascorbic acid stimulates this process. Dexamethasone was demonstrated to be less vital in this model of VSMC calcification. In vitro calcification of rat aortic VSMCs is not affected by amlodipine treatment, but is stimulated by atorvastatin treatment. As high concentrations of atorvastatin have a pro-apoptotic effect, the pro-calcification effect of atorvastatin may be explained by the production of apoptotic bodies that act as foci for calcium deposition. The latter finding may explain the plaque-stabilizing effect reported for statins.

calcification
vascular smooth muscle cells
calcium antagonist
statin
atherosclerosis
in vitro model


Growth hormone axis in chronic kidney disease

Chronic kidney disease (CKD) in children is associated with dramatic changes in the growth hormone (GH) and insulin-like growth factor (IGF-1) axis, resulting in growth retardation. Moderate-to-severe growth retardation in CKD is associated with increased morbidity and mortality. Renal failure is a state of GH resistance and not GH deficiency. Some mechanisms of GH resistance are: reduced density of GH receptors in target organs, impaired GH-activated post-receptor Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling, and reduced levels of free IGF-1 due to increased inhibitory IGF-binding proteins (IGFBPs). Treatment with recombinant human growth hormone (rhGH) has been proven to be safe and efficacious in children with CKD. Even though rhGH has been shown to improve catch-up growth and to allow the child to achieve normal adult height, the final adult height is still significantly below the genetic target. Growth retardation may persist after renal transplantation due to multiple factors, such as steroid use, decreased renal function and an abnormal GH–IGF1 axis. Those below age 6 years are the ones to benefit most from transplantation in demonstrating acceleration in linear growth. Newer treatment modalities targeting the GH resistance with recombinant human IGF-1 (rhIGF-1), recombinant human IGFBP3 (rhIGFBP3) and IGFBP displacers are under investigation and may prove to be more effective in treating growth failure in CKD.
Learning objectives:
To review the normal growth pattern in childhood and its alteration in chronic kidney disease.
To understand the mechanisms believed to be responsible for growth failure in chronic kidney disease.
To understand the new evidence supporting therapy for growth failure in chronic kidney disease.
To identify targets for future therapies for treatment of growth retardation in chronic kidney disease.
Introduction
Growth retardation is a common result of chronic kidney disease (CKD) in childhood. Children with CKD fail to achieve the final adult height consistent with their genetic potential [1]. Data from the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS) 2005 database revealed that 36.9% of children with CKD had statutory growth impairment. Even though the growth failure was correlated with the degree of renal impairment, those with mild reduction of glomerular filtration rate (GFR) also exhibited short stature. Children were short at initiation of dialysis, with younger age associated with more severe growth failure and little improvement in height standard deviation score (SDS). The mean height deficit for all renal failure patients was −1.85 (less than the third percentile of their healthy peers) at transplantation. This deficit was greater for male patients and for younger subjects. Those below age 6 years benefited most from transplantation in demonstrating acceleration in linear growth. For subjects aged 6–12 years, the linear growth remained stable, and those older than 12 years of age had no increase in their height deficit score [2–4].
Despite advances in medical care, growth failure in CKD is associated with increased morbidity and mortality. Furthet al. demonstrated from the United States Renal Data System (USRDS) database that patients with severe-to-moderate growth failure had increased hospitalization rates and increased risk of death [5].
Growth retardation is assessed by the SDS or height deficit score, which measures the patient’s height in comparison with that of unaffected children of similar age. Wong et al. demonstrated that, among pediatric patients on dialysis or after transplantation, each SDS decrease in height was associated with an increase in death by 14% [6].
Alteration of normal growth patterns in CKD
Linear growth is unique to childhood. Complex biological processes are responsible for maintaining normal growth.
Infancy is the fastest growing period of childhood; one-third of the total growth occurs in the child’s first 2 years of life, and this is mainly nutrient dependant. This growth is markedly decreased in congenital CKD, with the greatest height deficit occurring in the child’s first year of life, especially during the first 4 months [7]. Thus, the earlier the onset of kidney disease, the more severe the growth disturbance.
Additional factors affecting growth are inadequate protein and calorie intake, water and electrolyte losses in polyuric and salt wasting conditions, anemia, metabolic acidosis, renal osteodystrophy, and resistance to hormones mediating growth. Early nutritional intervention and prevention of metabolic deficits of renal failure can preclude the development of growth failure in this period. Conservative treatment (no use of growth hormone) in pre-dialysis patients from birth to 3 years, revealed a favorable growth rate, with height velocity at 22.2 cm/year, 10.9 cm/year and 7.6 cm/year for each year, respectively, all of which were higher than the lower two standard deviation scores [5]. Further discussion of these factors is beyond the scope of this article.
During the mid-childhood period, a constant growth rate of 5–7 cm/year is demonstrated, mainly under the control of growth hormone (GH) and thyroid hormone. Children with congenital CKD grow at the percentile achieved at the end of 2 years of life. Even though one of the most important predictors of growth impairment is the degree of renal insufficiency, significant short stature has been seen at all levels of renal function [3, 7].
At the onset of puberty, the growth hormone/insulin-like growth factor (GH/IGF-1) axis is activated by small increases in estrogen and testosterone in girls and boys, respectively. The onset of puberty is delayed in adolescents with CKD; peak height velocity is delayed by approximately 2.5 years. The pubertal growth spurt is delayed, shortened and associated with a reduced growth velocity. The pubertal height gain is about 65% of that seen in healthy children without CKD and is likely due to the shortened growth spurt [1]. These effects of delayed puberty in CKD are mediated by a loss of the normal pulsatile hypothalamic release of the gonadotropin-releasing hormone (GnRH).
GH and IGF axis
Growth hormone is the key endocrine regulator of postnatal growth (Fig. 1) [8]. The anterior pituitary secretes growth hormone in a pulsatile manner stimulated by the growth hormone releasing hormone (GHRH) and inhibited by somatostatin. GH mediates its somatotropic actions directly and mainly through IGF-1. Ghrelin, a growth hormone-releasing peptide expressed in the stomach and hypothalamus, is involved in hormonal and nutritional regulation of GH release [9].
Fig. 1GH/IGF-1 axis in CKD: deranged somatotropic axis in chronic renal failure. The GH/IGF-I axis in CRF is changed markedly, compared with the normal axis shown here. In CRF the total concentrations of the hormones in the GH/IGF-I axis are not reduced, but there is reduced effectiveness of endogenous GH and IGF-I, which probably plays a major role in reducing linear bone growth. The reduced effectiveness of endogenous IGF-I likely is due to decreased levels of free, bioactive IGF-I as levels of circulating inhibitory IGF-binding proteins (IGFBPs) are increased. In addition, less IGF-I is circulating in the complex with acid labile subunit (ALS) and IGFBP-3 as a result of increased proteolysis of IGFBP-3. Together, these lead to decreased IGF-I receptor activation and a decreased feedback to the hypothalamus and pituitary. Low free IGF-I and high IGFBP-1 and IGFBP-2 levels probably contribute to reduced renal function and lead to reduced stature. The direct effects of GH on bone, which are poorly understood, also are blunted. Reprinted from [8] with permission
Growth hormone and the IGF-1 axis play a major role in growth failure in CKD. Random fasting serum levels of GH are normal or increased in children and adults with CKD, depending on the extent of renal failure. The half-life of GH is prolonged, due to decreased metabolic clearance secondary to decreased functional renal mass in proportion to the degree of renal dysfunction. A high normal calculated GH secretion rate and amplified number of GH secretory bursts have been reported in pre-pubertal children with end-stage renal disease, likely due to attenuated IGF-1 feedback [8]. This has led to the concept of GH insensitivity or resistance in uremia.
Pubertal patients with advanced CKD have a reduced GH secretion rate, indicating altered sensitivity of somatotropic hormones to the stimulatory effects of sex steroids [10].
GH resistance
GH receptors One mechanism for GH resistance is a reduced density of GH receptors in target organs. Determination of the concentration of serum growth hormone binding protein (GHBP), which is a cleaved product of the GH receptor, may be used to assess GH receptor density in tissues, particularly liver, since GHBP is derived mainly, but not exclusively, from the liver. GHBP is low in children and adults with CKD and proportionate to the degree of renal dysfunction. Serum GHBP correlates with both spontaneous growth rate and response to GH therapy, and it is an indirect indicator of sensitivity to both exogenous and endogenous GH [11, 12]. However, there is controversy as to the reliability of serum GHBP level as a marker of GH receptor levels in specific tissues [13].
Janus kinase/signal transducer and activator of transcription signaling Another mechanism for GH resistance in uremia is a defect in post-receptor GH-activated Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling (Fig. 2) [14]. GH action is mediated by the binding of GH to the growth hormone receptor (GHR), resulting in its dimerization and the auto-phosphorylation of the tyrosine kinases, Janus kinase 2 (JAK2), which, in turn, stimulates phosphorylation of signaling proteins, STAT proteins STAT1, STAT3 and STAT5. Upon activation, these STAT proteins translocate to the nucleus and activate GH-regulated genes. An intact JAK2-STAT5b signaling pathway is essential for GH stimulation of IGF-1 gene expression. In uremia, a defect in the post-receptor GH-activated JAK2 signal transducer and STAT transduction is described as one of the mechanisms of GH resistance [14].
Fig. 2Growth hormone-mediated JAK/STAT signal transduction. GH activates several signaling pathways via JAK2, including the JAK/STAT pathway [22, 23]. Binding of GH to its receptor (GHR) activates JAK2, which then self-phosphorylates. This is followed by phosphorylation of the GHR and, subsequently, STAT 1a, STAT 3, STAT 5a, and STAT 5b, members of a larger family of cytoplasmic transcription factors. These phosphorylated STATs form dimers that enter the nucleus, where they bind to specific DNA sequences and activate their target genes, IGF-1 and some suppressors of cytokine signaling (SOCS). Deletion of STAT5 expression leads to retarded body growth, and STAT5b is required for GH-mediated IGF-1 gene expression. In renal failure phosphorylation of JAK2 and the downstream signaling molecules STAT5, STAT3, and STAT1 is impaired, as are the nuclear levels of phosphorylated STAT proteins. This important cause of uremic GH resistance may result, in part, from up-regulation of SOCS2 and SOCS3 expression with suppressed GH signaling and also from increased protein tyrosine phosphatase activity, with enhanced dephosphorylation and deactivation of the signaling proteins. Reprinted from [14] with permissionThe JAK2/STAT pathway is regulated, among other factors, by suppressor of cytokine signaling (SOCS) proteins, which is induced by GH. These proteins bind to JAK2 and inhibit STAT phosphorylation [15]. Up-regulation of SOCS has been described in inflammatory states and may play a similar role in CKD [16].
IGF and IGFBP Daughaday et al. proposed the somatomedin hypothesis: GH leads to an increase in IGF-1 (formerly called somatomedin) c) from the liver, which, in turn, reaches the growth plate to mediate its action [17]. However, it was later demonstrated that all the effects of GH are not mediated through IGF-1. Also, the liver is not the only source of IGF-1, and it is produced in other tissues. Circulating IGF-1 mainly derived from the liver acts as an endocrine hormone. IGF-1 produced locally acts as a paracrine/autocrine hormone. The majority of circulating IGF in the serum exists as a 150 kDa complex, including the IGF molecule, IGF binding protein 3 (IGFBP3) and the acid labile subunit (ALS). IGF-1 gene knockout in mice results in miniature mice that die soon after birth. Knock-out of IGF-1 in the liver alone results in relatively normal sized mice with a 75% reduction in circulating IGF-1 levels, suggesting that local IGF-1 may be more important for body growth than liver IGF-1 [18]. ALS knock-out mice also exhibit relatively normal growth and development, despite a 65% reduction in circulating IGF-1 levels. Disruption of both liver IGF-1 and ALS genes results in 85–90% reduction in circulating IGF-1 levels and significant reduction in linear growth, suggesting that a threshold concentration of IGF-1 is necessary for normal growth [19].IGF-1 is the predominant IGF during the rapid growth period of puberty. The levels of IGFs (IGF-1 and IGF-2) are normal in pre-terminal renal failure. In end-stage renal disease IGF-1 level is normal or decreased and levels of IGF binding proteins (IGFBP) are increased, thus resulting in a net decrease in IGF bioactivity. Free IGF-1 is decreased in proportion to renal failure [20, 21].IGF-1 is an anabolic hormone; it binds to the IGF-1 receptor (IGF-1R) at the α subunit, where it activates tyrosine kinase intrinsic to the IGF-1Rβ subunit. In experiments with rats with CKD, skeletal muscles show decreased serum levels of IGF-1 and IGF-1 mRNA, while IGF-1R mRNA and IGF-1R numbers are increased, with normal binding affinity. However, there is resistance to post-receptor signal transduction; both auto-phosphorylation of the IGF-1R tyrosine kinase and activity of the IGF-1R tyrosine kinase to the exogenous insulin receptor substrate-1 (IRS-1), a natural substrate for IGF-1 receptor tyrosine kinase, are diminished in skeletal muscle of CKD rats. These abnormalities may account for the resistance of IGF-1 to protein turnover in skeletal muscles in CKD [22].IGFs are transported in plasma bound to IGF binding proteins (IGFBPs), which are responsible for extending their half-life and controlling their bioavailability. There are six different IGFBPs, based on their respective amino acid sequence. Of the IGFs in the serum, 75% circulate as a ternary complex bound to IGFBP3 and another subunit, acid labile subunit (ALS), to form major 150 kDa complexes. This acts as a reservoir and cannot cross the endothelial barrier. Some 20–25% of IGFs are present as smaller binary complexes with other IGFBPs forming the minor complex (35 kDa) [23]. Approximately 97% of IGF is bound; 1% of IGF-1 is in a free bioactive form. In healthy children there is a 25% excess of IGFBPs over IGF, while, in pre-terminal renal failure, IGFBPs are 150% in excess of IGFs, and in end-stage renal disease they are 200% in excess, thus reducing the bioavailability of IGF [21].IGFBP3 is the most abundant of all the binding proteins. IGFBP3 and IGFBP5 are similar in many aspects; structurally, they are both closely related, both potentiate IGF action and are up-regulated by GH. The serum level of intact IGFBP3 is normal in CKD, while the fragmented fraction (29 kDa) is increased but has a reduced affinity for peptides [24]. IGFBP5 level is not altered, but it exists mostly in the fragmented form.Intact IGFBP1, IGFBP2, IGFBP4 and IGFBP6 are elevated in CKD in relation to the degree of renal dysfunction [25]. IGFBP1, IGFBP2 and IGFBP6 are growth inhibitors of IGF-dependant proliferation of chondrocytes. Increased levels of IGFBP1 and IGFBP2 have been shown to correlate negatively with height [26]. Ulinski et al. analyzed serum levels of IGFBP4 and IGFBP5 in 89 CKD patients. There was a fourfold increase in IGFBP4, while IGFBP5 was not significantly increased. IGFBP4 level was inversely correlated with glomerular filtration rate and standard height. IGFBP5 level was positively correlated with standard height [25].
Ghrelin
Ghrelin is a newly discovered, 28-amino acid peptide, with growth hormone releasing properties [growth hormone secretagogue (GHS)], expressed most abundantly in the stomach and also in smaller quantities by pancreatic islet cells and hypothalamus. Ghrelin levels are increased by fasting, cholinergic stimulation, estrogen and recombinant hIGF-1, and are decreased by food intake [27].
Ghrelin has been shown to increase the release of GH which is amplified by co-administration of GHRH, and acts at the level of the hypothalamus and pituitary. The role of ghrelin in the growth abnormalities in CKD has yet to be defined.
Treatment with recombinant human growth hormone
Studies in the past have shown that therapy with recombinant human growth hormone (rhGH) in CKD is safe and efficacious, and that it increases growth rate and improves standardized height [28, 29]. Haffner et al. studied the effect of growth hormone treatment on final adult height of children with CKD (Fig. 3) [1]. In contrast to the controls that had persistent growth failure, children treated with rhGH demonstrated sustained catch-up growth. In treated children the standardized height increased from baseline to a mean final adult height of 1.6 ± 1.2 SD below normal, while, in untreated control children, it decreased from baseline to a mean final adult height of 2.1 ± 1.2 SD below normal. Of the children treated with rhGH, 65% reached a final adult height within the normal range, although it was significantly below the genetic target [1].
Fig. 3Growth hormone treatment in chronic kidney disease: change from initially predicted adult height at baseline in 38 children (32 boys and six girls) with chronic renal failure who received growth hormone treatment compared with 50 control children with chronic renal failure who did not receive growth hormone, according to gender. Values are means ± SD. Asterisks indicate significant differences from the previous period (P < 0.001) and daggers indicate significant differences from the children who were not treated with growth hormone (P < 0.001). Reprinted from [1] with permission
Poor growth outcomes after renal transplantation are associated with corticosteroid use, persistent CKD and abnormalities in the GH-IGF-1 axis. The use of rhGH after transplantation leads to catch-up growth associated with increase in IGF-1 levels [26]. Using the NAPRTCS database of 513 renal transplant recipients receiving rhGH, Fine and Stablein demonstrated that final adult height was superior in rhGH treated patients compared to controls. Allograft function and graft failure rates were similar in both groups, with no increase in adverse events [30].
RhGH treatment in renal failure is associated with an elevation in serum free IGF-1 [31]. RhGH increases IGFBP3, IGFBP4, and IGFBP5, decreases IGFBP1 and has no effect on IGFBP2 and IGFBP6. RhGH also increases IGFBP4, but does not correlate with change in height SDS, since most of the IGFBP4 in serum is fragmented [25, 31]. RhGH-mediated increase in IGFBP3, IGFBP5 and ALS results in increased ternary complexes and correlates positively with the increment in height SDS [31].
Among the growth-promoting effects of treatment with rhGH in CKD are anabolic effects, demonstrated by increase in body weight and mid-arm muscle circumference. The recommended dose approved for treatment of growth failure in CKD is 0.35 mg/kg per week (28 IU/m2 per week) [3].
Targets for future therapy:
Treatment with recombinant IGF-1
The GH resistance associated with CKD may be amenable to treatment with recombinant human IGF-1 (rhIGF-1). In children with GH-receptor deficiency or GH-inactivating antibodies, rhIGF-1 treatment resulted in a modest increase in growth velocity and height SDS, although less than that expected with rhGH [32].
In addition, short-term administration of rhIGF-1 has been shown to increase glomerular filtration rate and renal plasma flow in patients with end-stage renal disease and in healthy subjects. Vijayan et al. demonstrated sustained improvement in renal function in a 31-day randomized, double-blinded, placebo-controlled trial of 15 patients with advanced CKD, who had received intermittent doses of rhIGF-1 [33].
One reason for the use of IGF-1 treatment in CKD is that while patients are GH sufficient, they are GH resistant. Therefore, rhIGF-1 may be more beneficial than GH as therapy in CKD and merits further investigation as an agent for treatment of growth failure and improvement in renal function in CKD [34].
Combined use of rhGH and rhIGF-1
Animal studies have shown that the combined use of rhGH and rhIGF-1 has an independent and additive effect. While whole-body growth in uremic rats was similar with treatment with either agent, an additive effect on longitudinal growth and anabolism was demonstrated when both agents were administered together [35].
Combined use of rhIGF-1 and rhIGFBP3
One of the concerns with the use of rhIGF-1 in children with normal GH production is that rhIGF-1 may suppress endogenous GH, IGFBP3 and ALS production, which may have a detrimental effect on growth. The other adverse effect of the use of rhIGF-1 observed in GH receptor-deficient patients is hypoglycemia, due to low levels of IGFBP3 and increased level of free IGF-1 available for binding to insulin receptors [32]. Both these effects can be reduced by the combined use of rhIGF-1 and rhIGFBP3 [36].
IGFBP displacers
At least two different IGF-1 analogs have been made, which have an affinity for IGFBPs but have no effect on IGF receptors, thus “displacing” IGF-1 from IGFBPs and elevating free IGF-1 levels. Animal studies in hypophysectomized rats and dwarf dw/dw rats treated with IGFBP displacers demonstrated increased kidney size, improved renal function, and the stimulation of weight gain and bone growth. This effect was enhanced when treatment with IGFBP displacers was combined with rhIGF-1 [37].
Conclusion
This review of the literature lends support to the concept that CKD is associated with GH “resistance”. Despite adequate treatment with rhGH and improvement in catch-up growth, children with CKD display a final adult height that is often below the genetic target. The potential for newer therapies with rhIGF-1, combined use of rhGH and rhIGF-1, combined use of rhIGF-1 and rhIGFBP3 or IGFBP displacers to improve both the short and long term outcomes in the treatment of the disturbances in the GH/IGF-1 axis in CKD awaits future investigations.

growth hormone
insulin-like growth factor
treatment
growth failure
chronic kidney failure


Penetrating spinal injury with wooden fragments causing cauda equina syndrome: case report and literature review

Study design: Case report Objective: To report an unusual case of cauda equina syndrome following penetrating injury to the lumbar spine by wooden fragments and to stress the importance of early magnetic resonance imaging (MRI) in similar cases. Summary of background data: A 22-year-old girl accidentally landed on wooden bannister and sustained a laceration to her back. She complained of back pain but had fully intact neurological function. The laceration in her back was explored and four large wooden pieces were removed. However 72 h later, she developed cauda equina syndrome. MRI demonstrated the presence of a foreign body between second and third lumbar spinal levels following which she underwent emergency decompressive laminectomy and the removal of the multiple wooden fragments that had penetrated the dura. Results: Post-operatively motor function in her lower limbs returned to normal but she continued to require a catheter for incontinence. At review 6 months later, she was mobilising independently but the incontinence remained unchanged. Conclusion: There are no reported cases in the literature of wooden fragments penetrating the dura from the back with or without the progression to cauda equina syndrome. The need for a high degree of suspicion and an early MRI scan to localise any embedded wooden fragments that may be separate from the site of laceration is emphasized even if initial neurology is intact.
Introduction
Penetrating injuries to the spine, although less common than blunt trauma from motor vehicle accidents, are important causes of injury to the spinal cord [1, 2, 8, 12, 13, 18]. They are essentially of two varieties—gunshot or stab wounds. Gunshot injuries to the spine are more commonly described and are associated with a higher incidence of neurological damage. On the contrary, the prognosis is better in stab wounds where surgery plays a greater role [9, 12, 14, 16, 18]. Very few case reports have been published on the onset of cauda equina syndrome (CES) following stab wounds. Injuries with sharp knifelike objects and rarely glass have been known to cause stab wounds to the spine [1, 10, 13, 14, 17]. However, there are no reports in the literature of penetrating injury by wood to the cord or cauda equina. We report a unique case of CES that developed in a patient almost 72 h following a penetrating injury to her back by a large wooden fragment.
Case history
A 22-year-old hairdresser accidentally slipped from the top of the stairs on to the wooden bannister which broke and as she landed at the bottom of the stairs, a piece of the wood caused a laceration to her back at the upper lumbar paraspinal region. She was taken to the nearby hospital where an open laceration to the area was found. She was complaining of severe back pain but had intact neurology in her legs. The wound was explored and debrided and four large pieces of wood were removed following which she was commenced on antibiotics. Her back pain persisted and 72 h later, she started complaining of incontinence with bilateral paraesthesia and pain down her legs. On examination, she had mildly reduced power (4/5) distally in both legs with reduced sensation in her perineum.
She was transferred to the Neurosurgical unit and a MRI scan of the thoracolumbar region (Fig. 1) was taken. This revealed an area of low signal intensity between the soft tissues extending towards the spinal canal into the conus at the level of the second and third lumbar spines (L 2 and L 3) with a high signal in the cord above. No obvious spinal haematoma was present. She underwent an emergency decompressive laminectomy of L 2 and L 3 with removal of several fragments of wood. At surgery, the spinous process and right hemilamina of L 2 were noted to be fractured and multiple wooden fragments were found to have entered the spinal canal (Fig. 2) penetrating the dura in the midline causing contusion and division of some of the nerve roots of the conus. These wooden pieces were carefully removed using the microscope, the dura repaired and a closed drainage system left in. Swabs for microbiological examination were obtained from the surgical site and post-operatively prophylactic intravenous cefuroxime 750 mg, thrice a day for the next 7 days were commenced. She was advised strict bed rest in supine position for 5 days while the drain was left in situ. Over this period of time, the pain and weakness in her legs resolved completely but she continued to have numbness in the perineal region and continued to require the urinary catheter. A repeat post-operative MRI scan demonstrated the high signal in the cord as before with no residual foreign body in the canal. At discharge the wound appeared satisfactory and she was mobilising independently. When reviewed 6 months later, she was otherwise asymptomatic apart from persistent altered sensation in her perineal area.
Fig. 1T 2 weighted Sagital MRI of the lumbar spine (left) demonstrates the wooden piece as an area of low signal intensity (arrow) within the soft tissues, extending into the spinal canal with an area of high signal in the cord above. T 2 weighted axial MRI (right) shows the wooden piece to have penetrated the theca on the left sideFig. 2Per-operative view (left) demonstrates the wooden piece within the spinal canal (arrows) with size of the multiple wooden fragments demonstrated (right)
Discussion
The most widely reported cause of penetrating injury to spine is gunshot injury [1, 2, 8, 12, 13, 15, 18] with relatively few case reports describing injuries by glass or knifelike objects [1, 10, 13, 17]. Wooden foreign bodies have been reported to penetrate the cranium, orbit, face and limbs [3, 6]. However to our knowledge, there are no reports in the literature of PI to the spine by wooden fragments with or without development of CES. The only remote resemblance to our case is that by Lunawat et al. [9] who reported the presence of tiny pieces of wood in the spinal canal of an 18-year-old man who presented insidiously with weakness in his legs and who had suffered a PI to his abdomen 6 years ago.
Our case is unique due to several reasons. It is the first report of a penetrating injury by wooden fragments into the lumar spinal canal producing CES. The mechanism of injury was the direct force of the sharp wooden fragment penetrating the upper lumbar paraspinal region. We believe that after piercing the skin and subcutaneous tissue, the piece of wood probably fragmented. While some of the fragments remained superficially in the deep subcutaneous tissue overlying the thoracolumbar fascia, others were pushed towards the spinous process and right hemilamina of L 2 which were fractured and subsequently the wooden fragments pierced the dura contusing the nerve roots of the conus. What is interesting is that she developed CES almost 72 h following the injury. Whether the fragments actually moved into the canal later as a result of her movement in bed following the initial wound debridement or whether she developed oedema at the site of nerve root injury is unknown.
We found MRI scan very useful to demonstrate and localise the foreign body and also to exclude any intra- or extradural haematoma or contusion in the cord or cauda equina. In our case some contusion was demonstrated in the cord (Fig. 1). Previous reports of penetrated wooden pieces into the neck or extraspinal areas of the body have commented on the risk of misinterpretation of CT appearance of wood which appears as an area of high attenuation [3, 6]. Wood is highly echogenic and shows clear acoustic shadowing on sonography [10]. MRI is a useful adjunct to both CT and sonography for the detection of non-metallic foreign bodies such as wood [15].
Surgery in PI is indicated for progressive neurological deficits, persistent cerebrospinal fluid (CSF) leak or for incomplete neurologic deficits with radiological evidence of compression [5]. However, surgery at the cauda equina, in such cases, may not be easy due to the dural tear associated with impingement of the foreign body with the contused nerve roots. In our patient urgent surgery was necessary as she developed progressive neurological deficit leading to CES with radiological evidence of a large foreign body causing thecal compression. Post-operatively, we took the extra precaution to prevent CSF leak from the wound by advising the patient a strict bed rest for 5 days. In our institute we advise 3–5 days of strict bed rest for patients with unintentional dural tears during Lumbar spinal surgery. This has been the practise by others too [19]. We do not routinely prescribe antibiotics for such cases. However, given the nature of this particular case, we decided to use prophylactic antibiotics in spite of negative bacteriological results. Incidentally it has been claimed that extraspinal sepsis is much more common than spinal (CSF) infection following PI to the spine [5]. We did not use methylprednisolone as there is no convincing evidence to suggest any advantage, and moreover there have been reports of myopathy following the administration of methylprednisolone in patients with acute spinal injury [7, 11].
Finally, neurological recovery from injury to the cauda equina is unpredictable and may be influenced by several factors [4] such as patient age, energy transfer to the neurovascular structures and timing of neural decompression, although the latter is debated [12, 14, 15]. Late motor recovery has been reported to occur after PI to the cauda equina up to 16 years after injury in one study [8]. Our patient had excellent symptomatic relief of her back and bilateral leg pain following surgery with resolution of the motor weakness in her legs. However, the perineal numbness has persisted and we would have to wait and see if this improves in future.
Conclusion
Penetrating wound to the upper lumbar spine is common with gunshot injuries but has never been described with wooden fragments. MRI scan provides excellent visualisation of the foreign body and also helps to exclude any underlying spinal haematoma or contusion. For all similar cases where a laceration containing wood is found, the authors recommend a high index of suspicion and a MRI scan must be performed early to look for separate fragments of wood deeper down. Neural decompression and removal of the foreign bodies should be performed to prevent neurological deterioration, infection and possible CSF leak.