MRI of the kidney—state of the art

Ultrasound and computed tomography (CT) are modalities of first choice in renal imaging. Until now, magnetic resonance imaging (MRI) has mainly been used as a problem-solving technique. MRI has the advantage of superior soft-tissue contrast, which provides a powerful tool in the detection and characterization of renal lesions. The MRI features of common and less common renal lesions are discussed as well as the evaluation of the spread of malignant lesions and preoperative assessment. MR urography technique and applications are discussed as well as the role of MRI in the evaluation of potential kidney donors. Furthermore the advances in functional MRI of the kidney are highlighted.
Introduction
The role of computed tomography (CT) and magnetic resonance imaging (MRI) in the evaluation of renal abnormalities is ever increasing. Although multidetector helical CT has taken the largest leap, MRI can be used in case of compromised renal function, severe contrast allergy, or in case radiation exposure is a problem, such as in children and pregnant women. Furthermore, MRI can be used as a problem-solving modality when the CT findings are nondiagnostic. Attempts are being made to use MRI for imaging of renal function, including perfusion [1, 2], glomerular filtration rate [1, 3, 4] and intrarenal oxygen measurement [1, 5].
MRI technique
In MRI of the kidneys, fast imaging techniques are essential because of respiratory motion of the kidneys [6]. When possible the scan should be performed within one breath-hold. The patient should get clear instructions on breath-hold technique. If the patient has difficulty with breath-holding, a short period of hyperventilation before breath-holding may be helpful. The scan should be performed during expiration because the kidney position is more constant in expiration than in inspiration. If the sequence is too long to perform in one breath-hold, respiratory triggering can be used [6]. Another technique of respiratory motion control is respiratory gating by use of a navigator pulse. In this technique the movement of the diaphragm is monitored by a very fast 1D MRI sequence. If breath-holding is not possible, signal averaging can be used, but the quality of the images will be limited. The use of a phased array body coil is preferable because of the improved signal-to-noise ratio. To prevent aliasing in coronal imaging, the patient’s arms should be raised above the head, or the arms may be supported by cushions, anterior to the coronal plane through the kidneys.
The imaging protocol for evaluation of the kidney at our institution on a 1.5-Tesla (T) MRI system consists of the following sequences:
Coronal T2-weighted half Fourier single-shot turbo spin echo sequence (HASTE) (TR infinite, TE 120 ms, flip angle 90°, breath-hold), serving as a localizer, but also supplying valuable T2-weighted information. The limitation of this sequence is a relatively low signal-to-noise ratio.Axial T2-weighted turbo spin echo sequence with fat suppression (TR 2,000 ms, TE 100 ms, flip angle 90°, respiratory triggering). This sequence provides for more detailed T2-weighted information. The T2-weighted sequence is especially helpful in characterizing cysts and intraparenchymal abscesses and in evaluating hydronephrosis. Furthermore, the T2-weighted sequence is helpful in detecting solid lesions.Axial T1-weighted gradient echo sequence, in-phase and opposed-phase (TR 180 ms, TE 2.3 ms/4.6 ms, flip angle 90°, breath-hold), preferably as a dual-echo sequence. Many solid renal lesions are hypointense compared to the renal parenchyma on T1-weighted images, but lesions with hemorrhage, lesions with macroscopic fat, melanin-containing lesions and cysts with high protein content may show hyperintense signal [6]. Opposed-phase T1-weighted gradient echo sequences can be used to prove the presence of small amounts of fat.Axial T1-weighted gradient echo sequence for dynamic imaging (TR 130 ms, TE 1.0 ms, flip angle 90°), using 30 ml intravenous gadolinium contrast, immediately followed by three breath-hold periods with four scan series per breath-hold. In this way pre-contrast and post-contrast images in arterial and nephrographic phase are obtained. Gadolinium-enhanced images are used for lesion detection and characterization.Coronal 3D fast gradient echo with fat suppression, obtained immediately after the dynamic series for delayed contrast-enhanced images (TR 3 ms, TE 2 ms, flip angle 15°). This sequence can be used for renal venous anatomy, for the analysis of (tumor) thrombus and for evaluation of extent of the tumor in the perinephric fat.
Currently 1- to 1.5-T systems are generally used for abdominal imaging, but the advent of 3-T MRI systems brings a twofold increase in the signal-to-noise ratio (SNR). The increase in SNR can be spent on higher resolution or on even faster imaging. When combined with parallel imaging techniques such as sensitivity encoding (SENSE), the speed of any sequence can be increased by up to a factor of four or higher. However, although 3-T MRI is promising, only a limited amount of research has been published on 3-T MR imaging for renal lesions, and its value has still to be established [7, 8].
Renal lesions
The main goal in the evaluation of renal lesions is to differentiate surgical lesions from nonsurgical lesions. Most simple cysts are easily recognized and don’t need further analysis. Complicated or multiloculated cysts need more attention in order to differentiate them from cystic carcinomas. In most solid renal lesions, neither CT nor MRI is able to reliably distinguish benign from malignant. Some solid lesions, however, may be identified as benign with high confidence, like angiomyolipomas. In general, if a lesion cannot be characterized as benign or malignant, it should be considered malignant [9].
Malignant renal lesions
Renal cell carcinomas
Renal cell carcinomas account for 3% of all malignancies in adults. Almost 50% are detected incidentally. As many as 85% of suspicious renal lesions are malignant [10]. Features indicating potential malignancy of a renal lesion are size of the lesion, the presence of calcifications, the distribution of the calcifications within the lesion, wall thickness and the presence of septa in case of a cystic lesion (Fig. 1), inhomogeneity of the lesion, extension of the tumor beyond Gerota’s fascia, and last but not least, enhancement after contrast administration. Concerning calcifications in cystic lesions, recent research suggests that the importance of these calcifications as a determinant of malignancy is relatively low [11].
Fig. 1Gradient echo images with intravenous gadolinium at baseline (a), 8 months later (b) and 15 months later (c). The complicated cortical cyst (arrow) in the left kidney on image (a) in a patient with Von Hippel Lindau disease progresses into a frank renal cell carcinoma with multiple enhancing internal septations (c). Several simple cysts are visible. Images courtesy of Roy S. Dwarkasing
Duchene found in 186 renal tumors that all tumors greater than 7 cm (n = 48) were malignant. About 80% of tumors smaller than 3 cm were malignant [10]. The differential diagnosis of solid renal lesions smaller than 7 cm consists of oncocytoma, angiomyolipoma, hemangioma, leiomyoma, and focal xanthogranulomatous pyelonephritis. Of these lesions, only cysts and angiomyolipomas can often be positively identified as benign lesions.
Generally, MRI is performed only after a renal lesion has been detected by ultrasound or CT. CT may be followed by MRI if the enhancement at CT imaging is indeterminate (10–20 Hounsfield Units) [12] or in case of suspected pseudo-enhancement. At CT examination, simple cysts may show pseudo-enhancement after intravenous contrast administration, which is an increase in attenuation of more than 10 Hounsfield Units, not caused by administered contrast or by partial volume effect but by technical factors [13].
Assessment of enhancement
The main MRI feature indicating potential malignancy of a renal tumor is enhancement after intravenous gadolinium administration, differentiating the lesion from a cyst. However, enhancement at MRI cannot be measured as easily as enhancement at CT. The MR signal is not calibrated, in contrast to density at CT imaging. The MRI signal depends not only on tissue characteristics, but also on the size of the patient, the gain setting of the MR system, the pulse sequence and the coils. The presence of enhancement can be assessed subjectively, by subtraction imaging, and by quantitative assessment. Subjective assessment of enhancement has been shown to be accurate in detecting renal cell carcinomas [14]. However, in a cystic lesion with only a small solid component, subjective assessment may be difficult and subtraction images may be used to better assess the presence of enhancement. It is important to realize that renal cell carcinomas may be hypovascular and therefore may show less enhancement than the surrounding renal parenchyma [15]. Also in these hypovascular lesions and in lesions that are hyperintense on T1-weighted imaging, subjective assessment may be difficult, and subtraction images may be of particular use [16].
Some investigators have studied quantitative assessment of enhancement by calculating the relative enhancement defined as the signal intensity increase after contrast administration compared to the signal intensity before contrast administration. Ho used relative signal intensity enhancement to differentiate cysts from malignant lesions. Using a threshold of 15% relative signal intensity enhancement after administration of intravenous gadolinium, Ho found in 74 patients with renal lesions a sensitivity of 100% and a specificity of 94% in the detection of renal cell carcinomas [17]. The relative enhancement peak was maximum between 2 and 4 min after injection of gadolinium contrast. It is worthwhile to note that cysts also showed a mean enhancement change of up to 5%. This pseudo-enhancement may be attributed to motion artifacts and volume averaging [14].
Subtypes of renal cell carcinoma
Several histological subtypes of renal cell carcinoma are recognized. The most frequent subtype is the conventional or clear cell carcinoma, which comprises 88% of all renal cell carcinomas, followed by papillary carcinoma (10%) and chromophobe carcinoma (2%). Collecting-duct carcinoma is very rare. The first three subtypes do not differ significantly in prognosis and show a 5-year survival rate of 73–88% [18]. Several attempts have been made to distinguish the subtypes of renal cell carcinoma by imaging features using CT characteristics [19, 20]. There are only a few studies using MRI to differentiate the subtypes of renal cell carcinoma. Outwater et al. described that clear cell carcinomas may show loss of signal intensity on opposed-phase images compared to in-phase images, due to intracellular lipid [21]. This effect is caused by the presence of fat and water protons in the same voxel, resulting in cancellation of the signal on the opposed-phased sequence. The intracellular lipid contributes to the histological appearance of the clear cell. Because oncocytoma and transitional cell carcinoma do not contain diffuse lipid, the loss of signal on opposed-phase images may allow differentiation between clear cell carcinoma on the one hand and oncocytoma and transitional cell carcinoma on the other hand. However, this distinction is of little clinical use. If the tumor does not show signal loss, it can still be a clear cell carcinoma [21].
Fat-containing renal cell carcinomas
Although rare, it has been claimed that renal cell carcinomas may contain macroscopic fat. Since intralesional fat has long been considered diagnostic for angiomyolipoma, these rare fat-containing renal cell carcinomas may easily be confused with angiomyolipoma. A few cases of fat-containing renal lesions, suggestive of angiomyolipoma, that appeared to be renal cell carcinoma have been reported [22–27]. With a few exceptions [25, 27], most of the reported cases also contained intratumoral calcifications [22–26], which is very rare in angiomyolipoma. Therefore, the presence of calcification in a lesion with macroscopic fat should be a warning that the lesion may very well be a carcinoma [28]. Because the accuracy of MRI in detecting calcifications is relatively low, it is important always to assess earlier ultrasound or CT examinations of the patient, which will likely have been made in most cases.
Less than 5% of renal cell carcinomas are cystic [9] (Fig. 2). On CT imaging, cystic renal lesions are classified using the Bosniak classification system [29]. This system was developed for CT, but recently its use has been evaluated in MRI by Israel et al. [12]. The CT and MRI findings in this study were similar in 81% of 69 renal masses. In the remaining 19% of cases, MRI showed more septa, increased wall thickness or increased enhancement compared to CT. This resulted in an upgrade of the Bosniak classification in seven cases (10%), of which two cases were upgraded to category 3 and two cases to category 4. The latter two cases appeared to be malignant at surgery. One of the cases upgraded to category 3 appeared to be benign, and the second one showed progression (patient refused surgery). The authors state that the Bosniak grading system is appropriate for use in MRI, but acknowledge that further research is necessary [12].
Fig. 2T2-weighted turbo spin echo sequence (a) showing a cystic multiloculated lesion in the left kidney (arrow) with high signal intensity content. The pre- and post-gadolinium images (b and c) show enhancement of the septations. Pathologic examination showed a cystic renal cell carcinoma
Staging
There are two staging systems for renal cell carcinoma, both based on the degree of tumor spread beyond the kidney. The Robson staging system [30] (Table 1) is still in use, but it is being replaced by the TNM staging system, developed by the American Joint Committee on Cancer (AJCC) [31] (Table 2). The TNM staging system is similar to the Robson system, but provides a more detailed description. Staging is usually performed using CT. Hallscheidt compared the performance of CT and MRI in the TNM system and found a similar accuracy in the staging of renal cell carcinoma [32].
Table 1Robson staging system for renal cell carcinoma [30]StageTumor extentITumor confined to the kidneyIITumor extension through the capsule of the kidney in the perirenal fat including the adrenal gland, no involvement of Gerota’s fasciaIIIaTumor extension into the renal vein or inferior vena cavaIIIbInvolvement of regional lymph node(s)IIIcInvolvement of regional lymph node(s) and extension into the renal vein or inferior vena cavaIVaTumor extension beyond Gerota’s fascia into adjacent organsIVbDistant metastasisTable 2AJCC TNM staging system for renal cell carcinoma (2002, sixth edition) [31]StageDescriptionTxNo information on primary tumor availableT0No evidence of primary tumorT1aTumor size 4 cm or less, limited to the kidneyT1bTumor size more than 4 cm but no more than 7 cm, limited to the kidneyT2Tumor size more than 7 cm, limited to the kidneyT3aTumor extension into the perinephric fat and/or renal sinus fat or the adrenal gland, but not beyond Gerota’s fasciaT3bTumor grossly extends into the renal vein or its segmental (muscle-containing) branches, or inferior vena cava below the diaphragmT3cTumor grossly extends into the inferior vena cava above the diaphragm or invasion of the IVC wallT4Tumor extension beyond Gerota’s fasciaNxNo information on regional lymph nodes availableN0No regional lymph node metastasisN1Metastasis in a single regional lymph nodeN2Metastasis in more than one regional lymph nodeMxNo information on distant metastases availableM0No distant metastasesM1Distant metastases
Determination of the extent of the tumor into the renal vein and the inferior vena cava (IVC) (TNM stage T3b and T3c) is important for the surgical approach. Involvement of the IVC is reported to occur in 4–10% of renal cell carcinoma patients [33]. If the tumor extends into the large veins, the upper level of the tumor thrombus dictates the surgical approach. If the extension is limited to the renal vein, it is necessary to know the distance to the IVC, in order not to dissect the renal vein through the intraluminal tumor. Extension into the IVC requires cavotomy with clamping of the IVC and the contralateral renal vein (Fig. 3). If a tumor thrombus extends above the hepatic veins, the liver should be mobilized to control the IVC above the hepatic veins. Extension into the right atrium generally requires cardiopulmonary bypass during surgery with hypothermic cardiac arrest [33].
Fig. 3T1-weighted gradient echo sequence after intravenous contrast of a large renal cell carcinoma in the upper pole of the left kidney with tumor thrombus extending into the IVC up to the level of the liver
For differentiation between bland thrombus and tumor thrombus, the use of gadolinium contrast is indicated, since the enhancement of the thrombus indicates a tumor, whereas the lack of enhancement indicates a clot [34]. Before the introduction of multidetector CT scanners, MRI was considered to be superior to CT in the assessment of intravenous tumor extension, especially at the level of the intrahepatic IVC. However, multislice helical CT scanners are nowadays able to generate multiplanar reconstructions in any direction at high resolution. Recent studies comparing multidetector CT (MDCT) and MRI showed no difference in accuracy of MDCT and MRI in assessing the extension of thrombus in the IVC [35, 36].
Invasion of the IVC wall (TNM stage T3c) is important to detect because in that case the surgeon has to partially resect and reconstruct the IVC. The accuracy of MRI in IVC wall invasion has not yet been well documented. One study reported a sensitivity of 100% and a specificity of 89% in the detection of IVC wall involvement, however, this study consisted of only 12 patients and no inclusion criteria were reported [33]. In this study it was noted that altered signal in the vessel wall and wall enhancement were nonspecific. The most reliable sign of IVC wall invasion was tumor signal both inside and outside the vessel wall [33].
The detection of lymph node metastasis by CT mainly relies on the size of the lymph nodes. In recent years, several studies have been published on the use of ultra-small superparamagnetic iron oxide particles (USPIO) as a negative contrast agent for the detection of small lymph node metastasis. The USPIO particles consist of an iron-oxide core covered with a low-molecular-weight dextran coating. After intravenous injection, the USPIO particles accumulate in healthy lymph nodes. The USPIOs are ingested by macrophages through phagocytosis and cause a decrease in signal intensity on T2- and T2*-weighted images. Gradient echo sequences are the most sensitive for these susceptibility effects. Lymph node metastases displace the macrophages in the lymph node and therefore do not show the loss in signal intensity seen in normal lymph nodes. To our knowledge no studies have been published on the use of USPIOs in renal cell carcinoma. However, a recent meta-analysis by Will et al. [37] describes the pooled results of several studies on USPIOs for several types of metastasis. In these studies, MRI with and without ferumoxtran, a first-generation USPIO, was compared with histology. MRI with ferumoxtran significantly improved the diagnostic precision compared to MRI without ferumoxtran. It was shown to be both sensitive and specific, especially in the detection of abdominal and pelvic metastasis.
MRI in nephron-sparing surgery
In recent years, interest in nephron-sparing surgery has been growing. At first, partial nephrectomy was mainly performed in case of a solitary kidney or diminished renal function, in order to preserve as much function as possible. Due to improving techniques and to increasing application of modern imaging modalities, the number of small, incidentally detected renal tumors is increasing [38]. This development has encouraged surgeons to use nephron-sparing surgery also for patients with normal renal function. The long-term follow-up data suggest that survival after partial nephrectomy of small renal cell carcinomas is comparable to total nephrectomy [39]. The ideal tumor for partial nephrectomy is smaller than 3 cm, is confined to the parenchyma of the kidney and has a peripheral location [40]. The presence of a pseudocapsule around a renal tumor is a sign of lack of perinephric fat invasion and therefore a favorable sign for partial nephrectomy [38, 41]. A pseudocapsule consists of compressed renal tissue and fibrous tissue. The sensitivity of CT in depicting a pseudocapsule is low (10–26%), whereas MRI shows a moderate to high sensitivity in depicting the pseudocapsule (54–93%) [41, 42]. On MRI, a pseudocapsule presents as a hypointense rim around the tumor on both T1-weighted and T2-weighted images, but can be best seen on the T2-weighted images and sometimes on gadolinium-enhanced GRE images [40].
Transitional cell carcinoma
Transitional cell carcinoma of the kidney is usually evaluated by intravenous urography, CT and endoscopy. However, MRI may play a role if CT and endoscopy are not feasible. CT may not be possible in case of poor renal function, and in case of ureteral obstruction, contrast excretion may be too limited to allow tumor detection. The ureter may not be accessible for endoscopy because of fibrosis and stricture of the ureter and ureter ostium. This may especially be the case in patients who have been treated for bladder cancer. These patients are at particularly increased risk for upper urinary tract transitional cell carcinoma [43]. Transitional cell carcinoma of the pyelum or ureter will generally show as an irregular mass projecting in the lumen. In the collecting system, transitional cell carcinoma is usually confined to the lumen (Fig. 4), but infiltrative growth into the renal parenchyma occurs and typically does not distort the renal contour. Chahal et al. applied MR urography (MRU) in 23 patients with high clinical suspicion of upper tract transitional carcinoma and hydronephrosis that could not be explained with other imaging modalities. MRU showed five renal pelvic transitional cell carcinomas and eight ureteral transitional cell carcinomas, confirmed by histology. In the remaining patients, no sign of transitional cell carcinoma was observed during 1-year follow-up [43]. Although these results are promising, the number of publications on MRI for transitional cell carcinoma is limited and more research is required to determine the value of MRI for the detection of transitional cell carcinoma.
Fig. 4T1-weighted gradient echo images before (a) and after (b) gadolinium administration. A mass in the renal pelvis (arrow) shows moderate enhancement after gadolinium administration. A transitional cell carcinoma was suspected, which was confirmed after nephrectomy
Benign renal lesions
Oncocytoma
Oncocytomas are benign, most often asymptomatic renal tumors. They represent 2–12% of renal masses. On MRI, oncocytomas show variable low signal intensity on T1-weighted images (Fig. 5) and heterogeneous high signal intensity on T2-weighted images. In 33–54% a central scar with low signal intensity on both T1- and T2-weighted images is visible. After contrast enhancement a spoke-wheel-like pattern may be observed [44]. Both a central scar and spoke-wheel pattern, however, may also be seen in renal cell carcinomas [38, 45] and are therefore not specific for oncocytomas. Oncocytomas may show a pseudocapsule, consisting of compressed renal parenchyma and fibrous tissue. One should be aware that a pseudocapsule is not specific for oncocytoma, since renal cell carcinomas can also be surrounded by a pseudocapsule. Because the characteristics of oncocytomas show considerable overlap with the characteristics of renal cell carcinomas, the therapy for a suspected oncocytoma is usually surgical [38].
Fig. 5Post-contrast fat-suppressed T1-weighted gradient echo images, arterial phase (a) and nephrographic phase (b) of a central oncocytoma (arrow). The tumor shows a hypointense central scar in the arterial phase. In the nephrographic phase, the central scar is slightly hyperintense
Angiomyolipoma
Angiomyolipomas are benign hamartomatous tumors, consisting of fat, smooth muscle and blood vessels. Angiomyolipomas are the only solid renal tumors that can be positively characterized using MRI [46]. Angiomyolipomas are identified by demonstrating macroscopic fat in the lesion (Fig. 6). The ability to differentiate angiomyolipomas is especially urgent in patients with tuberous sclerosis, since angiomyolipomas develop in about 80% of these patients, and at the same time these patients are at an increased risk of developing renal cell carcinomas. Macroscopic fat in a renal lesion can be detected by CT using density measurement and by MRI using fat-suppression techniques. On the opposed-phase gradient echo images, macroscopic fat is demonstrated by a hypointense rim surrounding the fat (India ink artifact) [46]. It must be noted that the India ink artifact also occurs at the interface between tumors that don’t contain fat and the surrounding perinephric fat, if the tumor extends into the perinephric fat.
Fig. 6T1-weighted gradient echo image (a), post-contrast fat-suppressed T1-weighted gradient echo image (b), and T2-weighted HASTE (half Fourier single-shot turbo spin echo) image of the left kidney (c). The hyperintense parts of the tumor in the lower pole (black arrow) on the T1-weighted image show a drop in signal intensity on the post-contrast fat-suppressed T1-weighted image, proving the presence of fat, while the hypointense parts of the tumor enhance after gadolinium. The fatty portions are hyperintense on the T2-weighted sequence, but not as high as the cyst (white arrow) in the midportion of the kidney. The MRI characteristics of the tumor in the lower pole are consistent with angiomyolipoma
If the amount of intralesional fat is small, the differentiation between angiomyolipoma and renal cell carcinoma may be difficult (Fig. 7). Kim et al. showed for CT that homogeneous enhancement and a prolonged enhancement pattern were significantly more prevalent in angiomyolipoma with minimal fat than in renal cell carcinoma [47]. Using both CT findings as a criterion for the differentiation of angiomyolipoma with minimal fat from renal cell carcinoma, they found a positive predictive value of 91% and a negative predictive value of 87%. It is likely that this is also the case in contrast-enhanced MRI, however this has not yet been proved.
Fig. 7Lesion in the upper pole of the right kidney in a patient with tuberous sclerosis (arrow). The lesion shows low signal intensity on T2-weighted turbo spin echo images (a) and intermediate signal intensity on in-phase T1-weighted gradient echo images (b). No signal loss is observed on the out-of-phase T1-weighted gradient sequence (c). The lesion shows moderate enhancement after intravenous gadolinium administration (d). Pathologic examination after resection of the lesion showed an angiomyolipoma. In this unusual case, no macroscopic fat was detected on MRI
It has been shown that clear cell carcinomas may show signal loss on opposed-phase images compared to in-phase images, due to intracellular lipid [21]. This loss of signal intensity should be distinguished from the signal loss at the interface of macroscopic fat and surrounding tissue in angomyolipomas on opposed-phase images, especially if the amount of macroscopic fat is small. If in doubt, the in-phase gradient echo images are often helpful because the fatty portion of angiomyolipomas will be hyperintense, whereas renal clear cell carcinomas are generally hypo- or isointense [6, 46]. Unfortunately, clear cell carcinomas are incidentally hyperintense on T1-weighted images [48]. In these cases spectral fat-suppression images should be used to prove the presence of macroscopic fat in the angiomyolipoma.
Attention should be paid to the possibility that carcinomas sometimes contain hemorrhage, causing high signal intensity on in-phase T1-weighted images. In these cases, opposed-phase images and spectral fat suppression will not show a drop in signal intensity.
Xanthogranulomatous pyelonephritis
Xanthogranulomatous pyelonephritis is a rare chronic pyelonephritis, which may result in severe renal impairment. It is most common in middle-aged women, but it may also occur in children. Often Proteus or E. coli species are involved and the pyelonephritis is often accompanied by calculi, most typically staghorn calculi. It may be accompanied by calyx obstruction and parenchymal abscesses. In xanthogranulomatous pyelonephritis, the affected renal parenchyma is replaced by lipid-laden macrophages, resulting in the typical yellow-gray appearance of the lesion at macroscopy. It may involve the whole kidney, or it may be focal. Especially when it is focal, xanthogranulomatous pyelonephritis may be mistaken for a renal carcinoma. On T1-weighted images, the solid component of the lesion may be isointense or hyperintense, which can be attributed to the fatty component. On T2-weighted images, the signal intensity of the solid component is isointense to slightly hypointense. The parenchymal cavities filled with fluid and pus show high signal intensity on T2-weighted images and low signal intensity in T1-weighted images, varying according to the protein concentration in the cavity [49, 50]. In xanthogranulomatous pyelonephritis, the perirenal fascia may be thickened and show enhancement after gadolinium administration [49]. The absence of hyperintense signal on T2-weighted images of the solid components may be helpful in the differentiation between xanthogranulomatous pyelonephritis and renal tumor [50].
MR urography
In MR urography, the pyelocalyceal system and the ureters are visualized using heavily T2-weighted images or T1-weighted images with gadolinium contrast. On the heavily T2-weighted images, the urine in the pyelocalyceal system and ureters is hyperintense because of its long T2 relaxation time, whereas the surrounding tissue is hypointense. The HASTE or single-shot fast spin echo (SSFSE) is very suitable for this purpose; they are very fast with sufficient in-plane resolution [51]. The thin sections can be used for detailed evaluation; a maximum intensity projection is useful for overview of the urinary tract. For T2-weighted MR urography, it is essential that the urinary tract is sufficiently filled with urine. Therefore it is often necessary to use a diuretic if the urinary tract is not dilated [51]. Hagspiel et al. evaluated whether MRI of the urinary system of potential renal donors was feasible without diuretic stimulation or compression and found sufficient visualization of the urinary collecting system in only 14% of the rapid acquisition with relaxation enhancement (RARE) urograms and in 26% of the gadolinium-enhanced 3D fast low-angle shot (FLASH) urograms [52].
In contrast-enhanced MR urography, intravenous gadolinium can be combined with a T1-weighted 3D gradient echo sequence. In case the patient is unable to hold his breath, a fast 3D GRE EPI sequence can be used that offers the additional advantage of reduced ghost artifacts caused by ureteral peristalsis [53]. However, the conventional GRE images provide better high resolution images compared to the EPI images [53]. In contrast-enhanced MR urography, the images are acquired in the excretory phase, typically 5–8 min after intravenous gadolinium injection [51]. Additional use of a diuretic is advisable to increase excretion and to dilute the excreted contrast: if the excreted gadolinium is too concentrated, the T2* effect may cause signal loss [51]. The accuracy of MR urography in assessing renal obstruction is similar to CT urography [54, 55]. MRI has the advantage that it is better able to detect perirenal edema as a secondary sign of obstruction [54]. In a study by Sudah et al., gadolinium-enhanced MR urography showed renal calculi with considerably higher sensitivity than T2-weighted MR urography [55]. On MR urography, a calculus appears as a signal void, which is nonspecific: blood clots, gas, sloughed papilla and tumors may also appear as a low signal within the bright signal of urine [51]. If a signal void is not clearly detached from the wall of the pelvis or ureter, additional T1-weighted and contrast-enhanced images are necessary to further characterize the lesion.
MRI of potential donor kidneys
Due to the increasing demand for donor kidneys and the relative shortage of cadaver kidneys, the importance of living donors is increasing. Nephrectomy is increasingly performed by laparoscopic surgery to keep the burden for the donor as low as possible. A thorough pre-operative evaluation of the donor kidney is essential to keep the risks as low as possible. Especially for endoscopic nephrectomy, it is important for the surgeon to be informed about the arterial and venous vasculature of the kidney, about the presence of accessory vessels and about abnormal vessel location, like extrahilar branching and retrocaval position of vessels. Moreover, the surgeon needs to be informed about the presence of an abnormal collecting system and the presence of cysts or tumors [56].
In the imaging protocol, special attention should be focused on arterial and venous imaging, in addition to the standard parenchymal imaging. For the arterial MR angiography, a 3D fast GRE with intravenous gadolinium after timing bolus can be used (TR 4.7 ms, TE 1.4 ms, flip angle 30°), with coronal thin-section reconstructions. A relatively large flip angle (up to 40°) can be used to minimize background signal around the high signal of the renal arteries [56]. The venous angiography sequence should follow the arterial angiography immediately. Due to the excretion of gadolinium by the kidneys, the concentration of gadolinium in the renal veins is lower than in the renal arteries, causing lower contrast of the veins compared to the background. A lower flip angle (15°) can be used to compensate for the lower gadolinium concentration, at the expense of more background signal [56]. The 3D dataset can be used for the reconstruction of thin 2D sections for detailed evaluation, as well as for maximum intensity projection (MIP) reconstructions of the vessels (Fig. 8).
Fig. 8Pre-operative imaging of potential donor kidneys. a Gradient echo image after intravenous contrast, arterial phase, TR 3 ms, TE 1 ms, slice thickness 2.2 mm, flip angle 27°, coronal. b Gradient echo image after intravenous contrast, nephrographic phase, TR 4 ms, TE 1 ms, slice thickness 2.2 mm, flip angle 15°, coronal. The lower flip angle in the depiction of the renal veins was chosen to compensate for the lower gadolinium concentration in the renal veins
Although the time–of-flight technique can be used to evaluate renal arteries, it is not recommended for the detection of accessory renal arteries of small caliber [57]. It may be used to clarify intraluminal filling defects potentially caused by flow artifacts on gadolinium-enhanced MR angiography [6]. Phase contrast imaging can be used for detection and grading of renal artery stenosis [58, 59] but has limitations in evaluating potential kidney donors. The problem of motion artifacts in phase contrast imaging caused by the long acquisition time can be overcome by using interleaved gradient echo-planar technique, shortening the acquisition time from minutes to about 30 s [59]. However, it is difficult to select the proper velocity encoding gradient [6], and tortuous venous anatomy and low flow limit the use of phase contrast MRI in potential kidney donors [56].
Most studies on the accuracy of MR angiography (MRA) in the evaluation of renal vessels compared to digital subtraction angiography and CT angiography show similar or even better results [60–63], although not all studies confirm these findings [64]. In a recent study on 111 MRA examinations for donor nephrectomy, nine accessory arteries were missed, requiring anastomosis in four arteries. Of 14 kidneys with more than one vein, only 4 were identified by MRA, requiring anastomosis in 1 case [65].
MRI for imaging of renal function
In recent years, progress has been made in the use of MRI for the evaluation of renal function. Renal disease often causes impairment of renal function. Measurement of renal function can be used as an indicator of severity of disease and can direct therapy. The most simple tests of renal function are serum creatinine and creatinine clearance. However, these tests do not provide information about the function of each individual kidney. This information can be important in case of a living renal kidney donor, prior to nephrectomy or in case of renal artery stenosis. Renal scintigraphy provides information about the function of each kidney, but this test lacks anatomic detail. CT has the disadvantage of ionizing radiation and the use of potentially nephrotoxic contrast, which generally isn’t a problem in healthy kidneys, but may cause deterioration of renal function in diseased kidneys. MRI has the potential to combine the functional and anatomic information about each kidney individually [66].
Perfusion
Measurement of renal perfusion may be a tool to assess the significance of renal artery stenosis and to assess ischemic nephropathy, and it may be used in renal transplant assessment. Several techniques have been studied to measure renal perfusion [1]. The maximum slope Gd-DTPA technique uses the maximum slope of the Gd-DTPA enhancement curve in relation to the maximum Gd-DTPA concentration in the aorta to calculate renal blood flow. The Gd-DTPA concentration in the aorta can be calculated using pre-contrast and post-contrast relaxation times measured in the aorta, assuming a linear relationship between gadolinium concentration and the inverse of the pre- and post-contrast T1 difference. Calibration of the signal intensity is performed using phantoms with increasing gadolinium concentration [67].
Furthermore, attempts have been made to assess renal perfusion by arterial spin labeling. To prevent leakage of the contrast medium into the extravascular space, albumin-bound contrast agent is used [68]. However, the different MRI renal perfusion techniques still need to be validated so more research is needed to assess the clinical usefulness.
Glomerular filtration rate
The glomerular filtration rate (GFR) is a parameter that is used to assess renal function. The serum creatinine level is a rough indicator of the glomerular filtration rate, it is easy and cheap to obtain, but it provides no information about each individual kidney. For the measurement of the GFR by MRI, several techniques have been investigated. The first technique used MR spectroscopy to measure the T1 relaxation times of urine and serum samples taken at intervals after intravenous gadolinium administration. Calculation of the glomerular filtration rate was based on the linear relationship of 1/T1 to the serial dilution measurements of the serum and urine samples [69]. This technique still had the disadvantage of measuring the GFR of both kidneys together.
Another MR technique assessing each individual kidney was developed by calculating the extraction fraction (EF) of Gd-DTPA, which is the difference between renal arterial and renal venous Gd-DTPA concentration, normalized to the arterial Gd-DTPA concentration:  The gadolinium concentrations can be calculated after measurement of the T1 relaxation time of arterial and venous blood before and after intravenous gadolinium administration, using the formula  where R is the relaxivity of gadolinium, and T1pre and T1post are the T1 relaxation times before and after intravenous gadolinium. The glomerular filtration rate can then be calculated according to the formula where RBF is renal blood flow, which can be measured by phase contrast flow quantification [3], and Hct is hematocrit [66].
An alternative MRI technique to measure glomerular filtration rate also uses contrast-enhanced dynamic MRI and is based on a the time-dependent concentrations of Gd-DTPA in the cortex and the medulla—a two compartment model [1]. The different techniques to measure glomerular filtration rate still need to be validated, and their role in clinical practice needs to be established.
Diffusion-weighted MRI
In diffusion-weighted MR imaging, the image contrast is influenced by the Brownian motion of water molecules. The signal intensity is high if water molecules are restricted in their motion, which can be caused by cell membranes or, in the case of free fluid, by high viscosity. The MR signal intensity is low if water molecules can diffuse freely. Diffusion-weighted imaging has found its place in neuroradiology, especially for the early detection of ischemic brain lesions. Diffusion MRI can also be used for fiber tract mapping in cerebral white matter, by measuring the directional components of the diffusion. Because diffusion-weighted images are inherently T2-weighted, the images are influenced by the T2-shine-through effect. This is the presence of high signal intensity in restricted water, caused by the T2 effect. To cope with this effect, the apparent diffusion coefficient (ADC) may be calculated from two images acquired with different gradient duration and amplitude (b-values) and used for ADC mapping.
In renal MR imaging, the role of diffusion MRI is not yet as clear as in neuro-imaging. Fukuda et al. showed that diffusion in the kidney is anisotropic, due to the radial orientation of the tubules in the pyramids and the blood vessels in the renal cortex [70] (Fig. 9). Diffusion-weighted MRI has been applied in patients with solid renal masses as well as in pyelonephritis and renal failure [71, 72]. ADC mapping showed differences between the lesions and normal tissue, but more research is needed to know whether diffusion-weighted MRI can actually help in characterizing different abnormalities. Diffusion-weighted imaging has been applied to differentiate between hydronephrosis and pyonephrosis. Chan et al. found in a limited group of patients that diffusion-weighted imaging showed a hypointense pyelocalyceal system in hydronephrosis and a hyperintense pyelocalyceal system in pyonephrosis [73]. The hyperintensity in pyonephrosis is thought to be due to the high viscosity of the pus, whereas the free-moving molecules in hydronephrosis cause low signal intensity. The results of diffusion-weighted MRI in the kidney are still preliminary, and more research should reveal the value of its clinical application.
Fig. 9Diffusion-weighted tensor image of the right kidney on a 3T system. The renal pyramids show lower signal intensity than the surrounding parenchyma because of the radial orientation of the tubules in the pyramids, restricting the Brownian motion of the water molecules to one direction
Conclusion
The role of MRI in renal imaging is still mainly in differentiating benign lesions versus malignant lesions in patients who cannot undergo CT scanning with intravenous iodinated contrast media, or in cases with nondiagnostic CT results. MRI and CT show comparable accuracy in detection and characterization of most renal lesions. MRI can have additional diagnostic value in the evaluation of lesions with minimal amounts of fat or with intracellular fat. Data suggest that MRI has a higher sensitivity in evaluating complicated cysts, however, the clinical implications still have to be studied. There is evidence to suggest that MRI has a higher accuracy than CT in the evaluation of early lymph node spread. MRI is a suitable tool in the preoperative work-up of potential kidney donors. Functional MRI of the kidney has not yet found broad clinical application, but it has great potential. Through the ongoing development of functional MRI techniques, we may expect an increasing role for functional MRI in the management of patients with renal disease.

magnetic resonance imaging
humans
kidney diseases
kidney neoplasms


Oral Health in Women During Preconception and Pregnancy: Implications for Birth Outcomes and Infant Oral Health

The mouth is an obvious portal of entry to the body, and oral health reflects and influences general health and well being. Maternal oral health has significant implications for birth outcomes and infant oral health. Maternal periodontal disease, that is, a chronic infection of the gingiva and supporting tooth structures, has been associated with preterm birth, development of preeclampsia, and delivery of a small-for-gestational age infant. Maternal oral flora is transmitted to the newborn infant, and increased cariogenic flora in the mother predisposes the infant to the development of caries. It is intriguing to consider preconception, pregnancy, or intrapartum treatment of oral health conditions as a mechanism to improve women's oral and general health, pregnancy outcomes, and their children's dental health. However, given the relationship between oral health and general health, oral health care should be a goal in its own right for all individuals. Regardless of the potential for improved oral health to improve pregnancy outcomes, public policies that support comprehensive dental services for vulnerable women of childbearing age should be expanded so that their own oral and general health is safeguarded and their children's risk of caries is reduced. Oral health promotion should include education of women and their health care providers ways to prevent oral disease from occurring, and referral for dental services when disease is present.
Introduction
A woman's preconception and pregnancy experience with the two most prevalent diseases of the mouth—periodontal disease and dental caries—not only influences her own oral health status but also may increase her risk of other diseases such as atherosclerosis [1–4], rheumatoid arthritis [5], and diabetes [6], impact pregnancy outcome [7–9], and her offspring's risk of developing early and severe dental caries [10–13]. Although largely preventable through evidence-based interventions, both periodontal disease and caries in women of childbearing age are highly prevalent, particularly among low-income women and members of racial and ethnic minority groups. In addition, both periodontal disease and caries are typically asymptomatic for long periods of time with only intermittent painful exacerbations. The combination of high prevalence, insufficient treatment rates, missed preventive opportunities, and intermittent symptoms led the US Surgeon General to publish a report in 2001 on oral health in America characterizing dental and oral disease as a “silent epidemic” [14]. Socioeconomic factors, lack of resources to pay for care, barriers to access to care, and lack of public understanding of the importance of oral health and effective self-care practices all represent underlying reasons cited for observed inadequacies in oral health.
An important oral condition affecting many pregnant women is periodontal disease. Periodontal disease is a destructive inflammatory condition of the gingiva and bone that supports teeth. It is most commonly associated with a gram-negative anaerobic infection of these structures. Fluid that bathes the tooth at the gingival margin, known as gingival crevicular fluid, often contains inflammatory mediators and oral pathogens associated with periodontal disease.
The mechanisms underlying this destructive process involve both direct tissue damage resulting from plaque bacterial products, and indirect damage through bacterial induction of the host inflammatory and immune responses [15]. Destructive periodontal disease affects up to 15% of the population of childbearing age, with a relatively high proportion of pregnant women demonstrating some degree of periodontal disease [7, 8, 16]. Advancing age, smoking, and diabetes are risk factors for the development of periodontal disease [17]. Whereas periodontal disease is a chronic, local oral infection, systemic inflammation may also occur [15].
The second oral disease important to women of childbearing age because of its maternal-child health associations is dental caries. Dental caries is the pathologic process by which teeth “decay” and develop “cavities.” It occurs when acid is produced at the tooth surface by cariogenic bacteria in the dental plaque that metabolize dietary carbohydrates. Acquisition of these cariogenic bacteria, dietary practices that govern the caries process, use of fluorides that dampen the caries process, and utilization of dental care all link mothers’ and children's experience with tooth decay through biological, behavioral, and social pathways.
Is maternal oral health linked to pregnancy outcome?
Preterm birth
Preterm birth, delivery at less than 37 weeks’ gestation, occurs in approximately 12% of all births [18, 19]. Prematurity is the leading cause of neonatal morbidity and mortality in non-anomalous infants [20]. There are numerous and heterogeneous factors associated with preterm birth, such as low maternal body mass index, maternal smoking, and maternal infections [21]. In 1996, Offenbacher and colleagues [7] first reported a potential association between maternal periodontal disease and delivery of a preterm/low birthweight infant. In a case-control study of 124 pregnant women, they observed that women who delivered at less than 37 weeks’ gestation or an infant <2500 g had significantly worse periodontal disease than control women. The adjusted odds ratio for delivery of a preterm, low birth weight infant was ∼7; these data led the authors to conclude that periodontal disease may represent a previously unrecognized and clinically significant risk factor for delivery of a preterm low birth weight infant [7]. Extrapolation from these data suggested that 18% of the preterm, low birth weight infants born annually might be attributable to periodontal disease, and thus account for a significant proportion of the $5.5 billion annual hospital costs associated with the care of preterm/low birthweight infants. In a subsequent case-control study, Dasanayake et al. studied 55 pairs of women. Logistic regression indicated that mothers with ‘healthy gingiva’ were at lower risk for low birth weight infants [22]. Women in both of these case-control studies were examined at the end of pregnancy or after delivery, which does not convincingly prove an antecedent exposure and thus causality. Despite this limitation, these early studies led to the hypothesis that periodontopathic bacteria, primarily Gram-negative anaerobes, may serve as a source for endotoxin and lipopolysaccharides, which then increases local inflammatory mediators including PGE2, and cytokines, and that this increases systemic inflammatory mediators that can then lead to preterm birth [23]. Additionally, Jeffcoat et al. [9] examined the relationship between maternal periodontal disease and spontaneous preterm birth among 1313 pregnant women, and found that moderate/severe maternal periodontal disease identified early in pregnancy was associated with an increased risk for spontaneous preterm birth, independent of other traditional risk factors [9].
Despite these compelling data, it is important to recognize that other studies have failed to demonstrate any association between maternal periodontal disease and preterm birth. In a case control study conducted in London, Davenport et al. [24] examined 236 infants born at <37 weeks’ gestation or <2500 g and compared them to a random sample of 507 control infants born at ≥38 weeks’ gestation and weighing ≥2500 g. The authors found no evidence for an association between delivery of a preterm, low birth weight infant and periodontal disease and somewhat surprisingly, found that deeper mean tooth pocket depths at delivery was associated with a reduction in the risk of delivery of a preterm, low birth weight infant [24]. The authors surmised that these discrepant findings might be due at least in part to racial differences in study populations. In a follow-up longitudinal study of 3738 women, Moore et al. [25] found no association between maternal periodontal disease and preterm birth. However, there was an increase in second trimester fetal loss rates among women with periodontal disease [25].
In an effort to better understand the possible mechanism behind the association between periodontal disease and preterm delivery, Offenbacher and colleagues [23] measured gingival crevicular levels of PGE2 and IL-1ß in 48 mothers who delivered preterm, low birth weight infants compared to control women and discovered that gingival crevicular fluid levels of PGE2 were significantly higher in case compared to control women. Furthermore, among the primiparous women delivering preterm, low birth weight infants, a significant inverse association was demonstrated between birthweight and gestational age and gingival crevicular PGE2 levels [23].
It is not yet clear whether the relationship between periodontal disease and adverse pregnancy outcomes is causal or is a surrogate for another maternal factor. As further evidence to support the concept that maternal oral health is important for normal pregnancy outcome, other investigators have examined the effect of antepartum treatment of periodontal disease on preterm birth risk. Three published studies of antepartum versus delayed (postpartum) treatment of maternal periodontal disease demonstrate promise for this intervention for preterm birth prevention. The effect of periodontal interventions on pregnancy outcome was assessed in a prospective study designed to examine the relationship between periodontal disease and preterm low birthweight infants in a cohort of young, minority, pregnant and postpartum women. Of 164 women for whom birth outcome data were available, 74 were subjected to oral prophylaxis during pregnancy, and 90 received no periodontal treatment. The preterm/low birthweight rate was lower among women who received periodontal treatment compared to those who did not (13.5% vs. 18.9%) [26]. Lopez et al. conducted a randomized clinical trial to assess the impact of periodontal treatment initiated during pregnancy versus delayed until postpartum on preterm low birthweight infant rates. The incidence of preterm/low birthweight infants in the antepartum treatment group was 1.8% (3/163) and in the delayed/postpartum group was 10.1% (19/188), (odds ratio [OR] 5.5, 95% confidence interval [CI] 1.7–18.2, P=0.001). Multivariable logistic regression analysis showed that periodontal disease was the strongest factor related to delivery of a preterm/low birthweight infant (OR 4.7, 95% CI 1.3–17.1) [27]. The data from these two studies suggest that treatment of periodontal disease during pregnancy could reduce preterm/low birthweight infant rates [26, 27].
In a pilot intervention trial designed to assess the feasibility of conducting a trial to determine whether treatment of periodontal disease reduces the risk of spontaneous preterm birth, Jeffcoat et al. found that among women at high risk for preterm birth and presence of periodontal disease, scaling and root planning therapy initiated during pregnancy is tolerated by pregnant women and may reduce spontaneous preterm birth [28].
Other adverse pregnancy outcomes
Preeclampsia is a hypertensive disorder of pregnancy responsible for significant, maternal and fetal morbidity and mortality. Preeclampsia affects up to 5% of pregnant women [29]. The etiology of preeclampsia remains elusive. The underlying pathology may be related to a generalized intravascular hyperinflammatory state [29]. Some investigators have hypothesized a potential role for maternal periodontal disease as a risk factor for preeclampsia. In a retrospective analysis of data collected as part of the Oral Conditions and Pregnancy Study, Boggess et al. reported that women were at higher risk for preeclampsia if they had severe periodontal disease at delivery (adjusted odds ratio 2.4, 95% confidence interval 1.1, 5.3), or if they had periodontal disease progression during pregnancy (adjusted odds ratio 2.1, 95% confidence interval 1.0, 4.4) [30]. In a case-control study, Canakci et al. found that pre-eclamptic patients were 3.5 (95% CI=1.1–11.9) times more likely to have periodontal disease than normotensive patients [P < 0.01) [31]. In a study of 30 pregnant women, significantly higher periodontal probing depth and clinical attachment level scores were found among preeclamptic women compared with non-preeclamptic women. Gingival crevicular fluid levels of PGE2, TNF-α, and IL-1β levels were all significantly higher in the preeclamptic group [32]. Further study on the maternal and fetal inflammatory responses to chronic oral infection and on placental pathology in women with periodontal disease is needed to determine whether the relationship between periodontal disease and preeclampsia is causal or simply associative. If the relationship between maternal periodontal disease and preeclampsia risk proves causal in nature, then prevention of periodontal disease before pregnancy or treatment of periodontal disease during pregnancy may represent a novel approachs to the prevention of preeclampsia.
Is maternal oral health linked to children's experience with tooth decay?
Cariogenic bacteria are typically acquired by young children through direct salivary transmission from their mothers [10]. Factors influencing transmission are the levels of these bacteria in maternal salivary reservoirs, frequency and efficiency of transmission, and the child's receptivity to implantation, which is largely diet dependent. Additional factors include timing of transmission, which is affected by the window of infectivity and the age of the child, and the composition and flow of the child's saliva. The earlier the transmission and the more caries-supportive the diet, the earlier and more substantial the transfer will be. For this reason, mothers who have themselves experienced extensive tooth decay and therefore most likely harbor high titers of mutans streptococci in their saliva will more effectively transmit this infection vertically, thereby putting their young children at elevated risk for early childhood caries. Although maternal cariogenic bacteria can be isolated in the pre-dentate infant's mouth [11], these organisms become established in the dental plaque on the tooth surface only after teeth first appear at around six months of age. Because oral flora tends to remain stable over time, a woman's cariogenic flora before and during pregnancy anticipates her flora during the child's first years of life as well as the likelihood of transmitting infection early to her offspring. The lag time between infection and expression of a discernable cavity in a tooth depends upon additional factors, including the frequency of simple carbohydrate exposure in a child's diet, oral hygiene, and exposure to fluorides. The evidence that caries is frequently established as a pathologic process in the mouths of very young children is strong, as 28% of US children, over 4 million toddlers and preschoolers, experience one or more frank cavities by ages 2–5 years [33].
Given the biological and behavioral pathways that govern intergenerational transmission of caries activity, disease management, and use of dental care, it is not surprising that disparities in dental caries among adults are mimicked among their children. As with adults, children of color and children of low-income families experience substantially more extensive and severe disease and less treatment than their peers without these risk factors [33]. Fortunately, despite the high prevalence of caries in women and children, this disease is readily preventable or manageable though early and regular dental care, exposure to fluoridated water, use of appropriate topical fluorides including those in toothpastes, application of sealants to primary teeth, and adoption of a health-promoting diet like that suggested in the Dietary Guidelines for Americans [34].
Is preconception preventive oral health care the answer?
It is intriguing to consider preconception, pregnancy, or intrapartum treatment of oral health conditions as a mechanism to improve women's oral and general health, pregnancy outcomes, and their children's dental health. Evidence is currently weakest for interventions that seek to reduce the incidence of preterm low birth weight through oral care. The mechanism of periodontal disease-associated adverse pregnancy outcomes is as yet unclear, and althoughit is hypothesized that if the ‘insult’ occurs early (either at conception or implantation) the risk is greater, no direct evidence to confirms that this is the case. However, given the strong relationship between oral health conditions and periodontal disease and general health and well-being, oral health care should be a goal in its own right for all individuals. If treatment of periodontal disease is going to impact pregnancy outcomes, then it is likely that the therapy will be of greatest benefit before or in very early pregnancy.
The science supporting interventions before, during, and after pregnancy to reduce caries transmission is much stronger. Educational and behavioral interventions that reduce caries activity through appropriate use of fluorides, dietary guidelines, chlorhexidine gels and varnishes, and xylitol [35], can reduce a woman's caries activity and salivary cariogenic flora, thereby improving her own oral health and, at the same time, also reducing the risk of transmission to her offspring. In two landmark Swedish studies [12, 13], children of mothers who had their cariogenic oral flora suppressed were less likely to experience cavities, more likely to develop cavities later if they were affected, and had fewer cavities than children of control mothers. Pregnancy is itself often regarded as an opportune time for anticipatory guidance and oral health education, and is a suitable time, particularly during the second trimester, for dental repair.
Access to oral health care during pregnancy
The CDC's Pregnancy Risk Assessment Monitoring System (PRAMS) reported that only 23–43% of pregnant women received dental care during their pregnancies [36, 37]—a rate only half to two-thirds of US women's overall use of dental services (67%) (14). The PRAMS data revealed that overall, pregnant women covered by Medicaid were 24%–53% less likely to obtain a dental visit during pregnancy than women who are privately insured. Similarly, women who initiated prenatal care later than the first trimester, who did not intend the pregnancy, and who were poor were also less likely to obtain care [36]. In contrast, the Behavioral Risk Factor Surveillance System (BRFSS) revealed that 70% of pregnant women in the years 1999 and 2002 had received a dental visit in the prior 12 months [38]. One possible explanation for the higher level reported by BRFSS is that it includes three or more months of pre-pregnancy time, during which dental care utilization would be expected to reflect the national norm for women. However, in contrast to recognized disparities in dental care utilization [33], race and ethnicity were not significantly associated with dental care during pregnancy in the BRFSS study. The authors suggest that, “the prevalence of dental visits … probably reflects factors such as prevailing attitudes toward dental care, provider availability and practice norms, and salient features of medical and dental care delivery within the state.” Overall, women covered by Medicaid were 24%–53% less likely to obtain a dental visit during pregnancy than women who are privately insured. Similarly, women who initiated prenatal care later than the first trimester, who did not intend the pregnancy, and who were poor were also less likely to obtain care.
An important additional consideration is that dentists are reportedly reluctant to provide care to pregnant women because of concern about possible risks [39]. Current practice typically limits non-urgent dental treatment of pregnant women to the second trimester, as there is concern about possible teratogenic consequences during the first trimester and about the woman's comfort in the dental chair during the third trimester. A single study relating antepartum dental radiography with full term low birth weight raised concern about the safety of dental care during pregnancy [40], but was criticized for its methodology. Neither professional associations nor government agencies have promulgated any authoritative guidance regarding dental care of pregnant women, although multi-center NIH clinical trials are underway that will determine the impact of dental care for periodontal disease during pregnancy on preterm low birth weight outcomes. Currently, the New York State Department of Health is developing “Guidelines for Prenatal Care, Oral Health, and Child Health Professionals” that promotes routine use of dental care during pregnancy.
Independent of pregnancy, the presence and source of dental insurance coverage is an important predictor of dental care utilization with publicly insured adults experiencing higher levels of oral diseases but less access to dental care. Medicaid is particularly significant to dental care of pregnant women as this program covers approximately 1/3rd of births in the US. However, states vary widely in adult Medicaid dental coverage, and at present only 7 jurisdictions providing comprehensive care to eligible adults. In contrast, low income pregnant women seeking dental services find themselves with no coverage in 8 states, coverage for only relief of pain or infection in 18 states or eligible for a limited range of services in 18 states. Three states (UT, LA, CA) have recently expanded dental benefits specifically to pregnant women in anticipation of reduced rates of unfavorable pregnancy outcomes. Pregnancy may be the only time that some low-income woman can readily obtain dental care as some state Medicaid programs provide adult dental coverage only to pregnant women or enhanced coverage during pregnancy.
Conclusions and future directions
Data are emerging to support a role for maternal periodontal disease as an infectious risk factor for preterm birth and other adverse outcomes of pregnancy. The prevalence of periodontal disease and the possibility of preterm birth prevention by treatment of oral infection make this a novel approach to improve the health and well being of our mothers and their soon-to-be born children. Further studies to better understand the mechanism of periodontal disease-associated preterm birth will enable us to tailor treatment to those women who might benefit the most.
Data on the relationship between maternal and child experience with dental caries is well established. Therefore, regardless of the potential for improved oral health to improve pregnancy outcomes, public policies that support comprehensive dental services for vulnerable women of childbearing age should be expanded, so not only their own oral and general health is safeguarded but also so that their children's risk of caries is reduced. Particularly if NIH trials confirm that treating pregnant women for periodontal disease reduces the incidence of unfavorable birth outcomes, the Centers for Medicare and Medicaid Services should build on its September 2004 coverage expansions for pregnant women by stimulating the states to similarly expand oral health services for pregnant women.
The power of prevention needs to be brought to bear, as both periodontal disease and caries are overwhelmingly preventable through well recognized strategies including regular and effective home care for periodontal disease and use of fluorides and sealants for caries. To the degree that pregnancy provides a “teachable moment” in self-care and future child-care, prenatal education should universally adopt an oral health component. This educational intervention should prioritize those mothers who have suffered significantly from dental caries so that they can learn to effectively prevent transfer of this disease to their children.
To be effective, oral health promotion must first seek to educate women and their health care providers about the importance of oral health and must promote an understanding of their ability to prevent and manage both periodontal disease and caries and to thereby limit the personal and intergenerational consequences of both conditions.


The exploratory behaviour of rats in the hole-board apparatus: Is head-dipping a valid measure of neophilia?

The exploratory behaviour of laboratory rodents is of interest within a number of areas of behavioural pharmacology. However, how best to measure exploratory behaviour in rodents remains a contentious issue. Many unconditioned tests, such as the open field, potentially confound general locomotor activity with exploration. The hole-board apparatus appears to avoid this confound, as head-dipping into holes in the floor is assumed to be a valid measure of the subject's attraction towards novelty (neophilia). This study aimed to investigate whether head-dipping should be considered a valid measure of neophilia by comparing performance of adult male and female Lister hooded rats on the hole-board task (a) over repeated sessions and (b) when novel objects were absent or present underneath the holes. The results show that head-dipping initially decreased across repeated exposures, while time spent in the aversive central area increased. No change in head-dipping was seen in response to objects being placed underneath the holes. Rather than being a measure of neophilia, these results support the hypothesis that head-dipping represents an escape response, which declines as the subject becomes less fearful. These results are compared with previous studies of repeated exposure to other novel environments.
1
Introduction
When faced with an unfamiliar environment or object, animals often exhibit behaviour patterns that broadly can be termed exploration, such as locomoting around the environment, orientating towards novelty, and touching or sniffing novel objects (Berlyne, 1950, 1960; Glickman and Sroges, 1966; Welker, 1957). Exploration potentially provides an animal with new information about food sources, shelters or mating opportunities. However, by entering a new environment or attending to a novel stimulus, an animal might also increase it's risk of predation, aggression from conspecifics or other hazards. Whether an animal investigates or avoids novelty has been described as the outcome of an approach–avoidance conflict (Montgomery, 1954, 1955; Montgomery and Monkman, 1955) or as a balance between neophilic and neophobic tendencies (Greenberg, 2003). In motivational terms, neophilia can be defined as the attraction that an animal displays towards an object or place simply because it is novel, while neophobia is the aversion that an animal shows towards approaching a novel object or place (Greenberg, 2003). In behavioural terms, neophilia and neophobia can be considered respectively as curiosity-based approach to, and fear-based avoidance of, a novel stimulus (Hughes, 2007).
The exploratory behaviour of rodents has gained recent interest within a number of areas of behavioural pharmacology. For instance, researchers studying drug addiction are interested in the neural mechanisms underlying neophilia due to the apparent overlap with the neural mechanisms involved in the rewarding effects of drug-taking (Bardo et al., 1996). However, considerable controversy still surrounds the question of how best to measure neophilic and neophobic responses in laboratory animals. One of the most commonly used behavioural tests for laboratory rodents is known as the open field. Originally, the open field apparatus consisted of a flat, raised platform (Hall, 1934, 1936), although the term open field is now commonly used to refer to any enclosed arena that can range in size from a small box to a large playing field (Crawley, 1985; Whishaw et al., 2006). In such an arena, the overall level of locomotion and time spent in the centre of the arena (which is assumed to be aversive to rodents) are often interpreted as measures of exploratory behaviour.
However, some researchers have argued that forcing an animal to be in an enclosed area, or on an open platform, does not allow the animal to exhibit its ‘motivation’ to explore an unknown environment, as the task evokes a strong fear response (Birke and Sadler, 1986; Denenberg, 1969; Renner, 1990; Walsh and Cummins, 1976). Corticosterone levels have been found to rise in rodents on exposure to a novel open field environment (e.g. Marin et al., 2007; Matzel et al., 2006; Rees et al., 2006), and open field behaviour is influenced by some anxiolytic (anxiety-reducing) substances (Prut and Belzung, 2003). In particular, benzodiazepines and serotonin receptor agonists, which have anxiolytic effects in human beings, generally increase the proportion of entries into the centre of the open field in rodents (Prut and Belzung, 2003). Together, these data suggest that the open field may provide valid behavioural measures of fearfulness, but may have limited value for researchers interested in measuring neophilia. Another common concern with interpreting open field tests is that differences in the performance of animals in the open field may result simply from differences in overall locomotor activity, which could be unrelated to differences in exploratory behaviour (Berlyne, 1960; Birke and Archer, 1983).
In the 1970s, researchers began to use the hole-board apparatus, which consists of an enclosed arena with holes in the floor into which an animal can poke it's head, referred to as head-dipping (e.g. File and Wardill, 1975a,b; Nolan and Parkes, 1973). The frequency and duration of head-dipping are assumed to provide measures of neophilia (or directed exploration) that are independent from the general locomotor activity of the animal (File and Wardill, 1975a; Ljungberg and Ungerstedt, 1976). This apparatus has been argued, therefore, to avoid the difficulties of interpreting general locomotion that prove problematic in the open field, and a number of studies have shown that head-dipping and locomotion can vary independently of each other (e.g. Abel, 1995; Durcan and Lister, 1989; File, 1977; Lister, 1987; Rogers et al., 1999). In general, high levels of head-dipping are interpreted as indicative of neophilia, while low levels are assumed to result from a lack of neophilia or are assumed to reflect a high anxiety-like state in the animal (Crawley, 1985; Takeda et al., 1998). The hole-board task is currently being used as a test of neophilia in many areas of behavioural pharmacology (Kliethermes and Crabbe, 2006a).
Researchers have attempted to validate head-dipping as a measure of neophilia by administering different classes of drugs and by comparing different genetic strains of rodents in their performance on the hole-board task. For example, if head-dipping is a neophilic response that is suppressed by an anxiety-like response, treatment with anxiolytic agents is predicted to increase head-dipping. Such studies have produced conflicting evidence; for instances, treatment of rodents with anxiolytic benzodiazepines has been reported to increase (rats: File, 1977; mice: Nolan and Parkes, 1973; Takeda et al., 1998), decrease (rats: Pellow et al., 1985) or have no effect (rats: Sayin et al., 1992) on the frequency of head-dipping. A recent review has suggested that the effects of anxiolytic compounds on head-dipping behaviour are generally confounded by changes in overall locomotion, despite the claims that head-dipping is unrelated to locomotor activity (Kliethermes and Crabbe, 2006a). Similarly, a study of several inbred mouse strains reported that head-dipping and locomotion are highly correlated (Kliethermes and Crabbe, 2006b). Whether head-dipping can be interpreted as a valid measure of neophilia remains unresolved (Bilkei-Gorzó and Gyertyán, 1996; Renner, 1990).
The aim of this study was to investigate whether head-dipping behaviour should be considered a valid measure of neophilia by comparing performance of rats on the hole-board task (a) over repeated sessions and (b) when objects are placed underneath the holes. Repeated exposure to a novel apparatus is expected to produce a reduction in exploration as the animal becomes familiar with the environment, a process commonly referred to as habituation (Leussis and Bolivar, 2006). If head-dipping behaviour is a measure of neophilia, the frequency of head-dipping is therefore predicted to decrease over repeated sessions. In early study by Nolan and Parkes (1973), head-dipping by young mice (21–25 postnatal days) was reported to be lower on a second exposure to the hole-board apparatus compared to the first exposure. Two recent studies have also provided evidence that head-dipping by mice and rats decreases on repeated exposure to the hole-board apparatus (Gagliano et al., 2008; Mayeux-Portas et al., 2000). If this behaviour is a valid measure of neophilia, head-dipping is also predicted to be higher in the presence, than in the absence, of objects. Although an early study reported that rodents head-dip more frequently when objects are present (File and Wardill, 1975a), this finding has not received recent replication. Given that male and female rodents are reported to exhibit behavioural differences on the hole-board task (e.g. Aguilar et al., 2003; Ray and Hansen, 2004), subjects of both sexes were included.
2
Materials and methods
2.1
Subjects and housing
The subjects of this experiment were eight male and eight female adult Lister Hooded rats (supplied by Harlan, U.K.). The animals were housed in a single room, which was controlled for temperature and humidity and was maintained on a 12-h light:dark cycle (lights on at 07:00). The animals were housed in same-sex pairs in plastic and wire mesh home-cages (measuring 25 cm × 45 cm × 15 cm) with ad libitum access to rodent pellets and water. All guidelines and requirements set out in the Principles of Laboratory Animal Care (National Institutes of Health, U.S.A., Publication No. 86-23, revised 1985) and the U.K. Animals (Scientific Procedures) Act 1986 were followed.
2.2
Apparatus and experimental design
The hole-board apparatus consisted of a wooden, grey box, measuring 68 cm × 68 cm. The walls were 40 cm high, and the box was raised 28 cm above the ground on a metal stand. Four holes (4 cm in diameter) were cut into the floor of the apparatus; each hole was 28 cm from a corner of the box along the diagonal from the corner to the centre. The floor of the box was marked out into four outer areas and one central area using black masking tape. The central area was delineated by four lines of tape each 20 cm from one of the walls, while the four outer areas were marked out by diagonal lines of tape running from the corners of the floor to the corners of the central square. The four holes were thus located at the corners of the central square. The apparatus was located in a small testing room with dimmed white lighting. The stand of the apparatus was open on all sides, allowing the floor or objects to be dimly lit.
Each subject was tested ten times in the hole-board apparatus, once per day during two sets of five consecutive days (Monday–Friday and the following Monday–Friday). During the first set of five trials, no objects were present underneath the holes of the apparatus; during the second set of five trials, an object was placed on the floor under each of the four holes prior to the start of the trial, approximately 20 cm below each hole. The objects were all distinct from each other but were similar in size (approximately 10 cm in length or diameter: a black-and-white rubber ball, a purple plastic star, a red-and-white rubber pet toy, and a yellow, rubber dumb-bell shaped pet toy).
All trials were carried out between 09:00 and 17:00 h, and trials on males and females were alternated throughout the day. At the beginning of each trial, a subject was placed in one corner of the apparatus (always the corner closest to the door of the room), facing the centre of the arena. Each trial lasted 10 min. At the end of the trial, the subject was immediately placed into a carrying box and returned to the home cage. Between each trial, the floor and walls of the apparatus and the novel objects, if present, were cleaned with 70% alcohol solution.
2.3
Behavioural measurements
During each 10-min trial, behavioural data were recorded by the observer (C.N.) onto a spreadsheet that was divided into 60 10-s time blocks. Inter-observer reliability between two independent observers (C.N. and G.R.B.) was over 80%. The following behaviour patterns were recorded:(i)enter a new area: the animal moves from one area of the open field to another (all four paws had to be placed on the floor of a new area);(ii)head-dip: the animal places it's head into one of the holes, to a minimum depth such that the ears were level with the floor of the apparatus (a new bout of head-dipping was recorded if the animal raised it's head fully out of the hole before resuming);(iii)rear: the animal is stationary on it's backpaws and raises it's forepaws off the ground, extending it's body vertically.
The data on entries into a new area were used to calculate the total amount of locomotion (number of entries into all areas summed together) and the percentage of entries that were in made into the central area. The location of the animal during each of the 10-s time intervals was used to estimate the percentage of time spent in the central area.
2.4
Statistical analyses
The data were analysed using repeated-measure ANOVAs, with ‘objects’ (with or without) as a within-subject variable, ‘trial’ (trials 1–5) as a within-subject repeated measure and ‘sex’ (male or female) as a between-subject variable. Significant interactions were analysed further using simple effects post hoc tests (Howell, 2007).
3
Results
3.1
Total locomotion
The total amount of locomotion did not vary across trials (F4,56 = 0.096, n.s.) or vary with the presence or absence of objects (F4,56 = 0.071, n.s.). Females locomoted more on average than males (F1,14 = 4.871, p = 0.045; Table 1). There were no interactions between trial and sex (F4,56 = 1.433, n.s.), trial and object (F4,56 = 2.615, n.s.) or object and sex (F4,56 = 0.559, n.s.), and the three-way interaction between these variables was not significant (F4,56 = 1.174, n.s.).
3.2
Locomotion into the central area
The percentage of entries that were into the central area differed significantly across trials (F4,56 = 14.842, p < 0.001) and varied with the presence or absence of objects (F1,56 = 84.240, p < 0.001). There was also a significant interaction between trials and the presence or absence of objects (F4,56 = 7.662, p < 0.001). Post hoc analyses indicated that the proportion of entries into the centre increased over the first five trials and remained steady thereafter (Fig. 1a). The percentage of entries into the central area varied between the sexes (F1,14 = 5.209, p = 0.039), with females exhibiting an overall greater proportion of entries into the centre than males (Table 1). There were no interactions between trial and sex (F4,56 = 0.761, n.s.) or object and sex (F4,56 = 1.956, n.s.), and the three-way interaction between these variables was not significant (F4,56 = 2.567, n.s.).
3.3
Time spent in the central area
The percentage of time spent in the central area differed significantly across trials (F4,56 = 8.085, p < 0.001) and varied with the presence or absence of objects (F1,56 = 35.393, p < 0.001). There was also a significant interactions between trials and the presence or absence of objects (F4,56 = 3.493, p = 0.013). Further analyses indicated that the time spent in the centre increased over the first five trials and remained steady thereafter (Fig. 1b). The percentage of time spent in the central area varied between the sexes (F1,14 = 5.032, p = 0.042), with females exhibiting an overall greater percentage of time in the centre than males (Table 1). There were no interactions between trial and sex (F4,56 = 0.083, n.s.) or object and sex (F4,56 = 0.076, n.s.), and the three-way interaction between these variables was not significant (F4,56 = 1.001, n.s.).
3.4
Frequency of head-dipping
The frequency of head-dipping differed significantly across trials (F4,56 = 2.626, p = 0.043), and there was a significant interaction between trials and the presence or absence of objects (F4,56 = 4.482, p = 0.013). Further analyses indicate that head-dipping decreased over the first five trials and slightly increased towards the end of the experiment (Fig. 2). There was no main effect of the presence or absence of objects (F1,14 = 0.142, n.s.). The frequency of head-dipping varied significantly between the sexes (F1,14 = 15.401, p = 0.002), with females exhibiting an overall greater frequency of head-dipping than males (Table 1). There were no interactions between trial and sex (F4,56 = 0.324, n.s.) or object and sex (F4,56 = 0.553, n.s.), or a three-way interaction between these variables (F4,56 = 0.779, n.s.).
3.5
Frequency of rearing
The frequency of rearing differed significantly across trials (F4,56 = 3.204, p = 0.019), with frequency increasing slightly across sessions (Fig. 3). There was no main effect of the presence or absence of objects (F1,14 = 3.898, n.s.) or sex (F1,14 = 1.332, n.s.; Table 1). There were no interactions between trials and the presence or absence of objects (F4,56 = 0.457, n.s.), between trial and sex (F4,56 = 0.111, n.s.) or object and sex (F4,56 = 0.006, n.s.), or a three-way interaction between these variables (F4,56 = 1.708, n.s.).
4
Discussion
The aim of this study was to investigate whether head-dipping behaviour should be considered a valid measure of neophilia by comparing performance on the hole-board task (a) over repeated sessions (trials 1–10) and (b) when no objects were present (trials 1–5) and when objects were placed underneath the holes (trials 6–10). The results show that head-dipping was high during the first test, decreased over the following two trials and remained relatively stable during the rest of the experiment. The initial drop in head-dipping following the first trial could be interpreted in two ways. First, head-dipping could be indicative of a neophilic response that decreases as the animal becomes familiar with the apparatus, i.e. head-dipping represents directed exploratory behaviour that drops as the apparatus loses its novelty. If this interpretation is correct, we would also predict that head-dipping would be greater in the presence of objects; however, there was no evidence of an increase in head-dipping behaviour when objects were present underneath the holes. These results do not support the hypothesis that head-dipping is a valid measure of neophilia.
The second interpretation of the initial drop in head-dipping frequency is that head-dipping could represent a fearful, neophobic response, such that, on first exposure to the apparatus, the animal actively attempts to find an escape route (Renner, 1990). Adult male rats have been shown to exhibit an increase in circulating corticosteroid levels following a single exposure to the hole-board apparatus (Márquez et al., 2005, 2006), suggesting that testing in this apparatus is a stressful event. If this interpretation of head-dipping is correct, we would also predict that, as head-dipping behaviour declines, fearfulness would also decline. In favour of this interpretation, while head-dipping frequency declined over the first few tests, the amount of locomotion into the central area of the hole-board, and the time spent in the central area, greatly increased over these trials. Therefore, as fearfulness apparently decreased, head-dipping also decreased. If we assume that the fear experienced on exposure to a novel apparatus can be equated to normal or ‘state’ anxiety (Belzung and Griebel, 2001), these results contradict the assumption that head-dipping behaviour is suppressed by an anxiety-like response, in which case we might have expected head-dipping to vary in the opposite direction to anxiety-like behaviour.
A small number of previous studies on rodents have also presented evidence that the frequency of head-dipping decreases on second exposure to the hole-board apparatus (Gagliano et al., 2008; Mayeux-Portas et al., 2000; Nolan and Parkes, 1973). The early study by Nolan and Parkes (1973) investigated whether previous experience with the hole-board apparatus with a solid floor inserted would influence frequency of head-dipping on second exposure to the apparatus. The results indicated that prior exposure to the plain board reduced the frequency of head-dipping on subsequent exposure as much as prior exposure to the board with holes (Nolan and Parkes, 1973). Again, these results do not support the idea that head-dipping provides a valid measure of neophilia, rather than a neophobic response induced by an unfamiliar test situation.
In our study, head-dipping did increase in frequency towards the end of the experiment, after eight or more exposures to the apparatus. Therefore, we cannot reject the possibility that, as the subjects became very familiar with the apparatus, they engaged in a greater level of visual exploration through the holes. Rearing behaviour is also commonly interpreted as an activity by which an animal obtains information about distal environmental cues (Lever et al., 2006). Given that rearing behaviour gradually increased in frequency across the sessions, as the subjects presumably became less fearful, the amount of visual assessment of the testing room, both above and below the apparatus, may have increased. This interpretation is supported by an experiment carried out by Bilkei-Gorzó and Gyertyán (1996). This study reported that the effects of the anxiolytic benzodiazepine chlordiazepoxide on head-dipping behaviour in rats varied with the light intensity during testing, such that treatment decreased head-dipping in very bright light and increased head-dipping under normal light. The authors argued that head-dipping during the aversive testing condition (bright light) represents an attempt by the subject to find an escape route from the apparatus, while head-dipping in less aversive conditions represents visual exploration of the apparatus. Future studies could investigate whether head-dipping increases with extended familiarisation to the apparatus even in the absence of novel objects, and whether head-dipping increases when novel objects are brought closer to the holes, when new novel objects are placed under the holes for each test, or when objects are re-located beneath different holes.
Our results show that repeated exposure of subjects to a novel hole-board apparatus greatly affects the behavioural response, and that the neophobic response experienced by subjects during the first exposure to an apparatus apparently declines over further exposures. Repeated exposure to another commonly used behavioural test, the elevated maze, has been reported to result in a decrease in total locomotion and a decrease in the amount of time spent on aversive open sections of the maze (e.g. Cook et al., 2002; Dawson et al., 1994; Rodgers et al., 1996). In contrast, in our study, no change of overall locomotion was found on repeated exposure to the hole-board, and the amount of time spent in the aversive central area of the hole-board apparatus increased over sessions. The reason for the differences in the behavioural effects of repeated exposure to these two types of novel environments may be related to differences in the design of the two types of apparatus. Repeated exposure to an elevated maze, which consists of open areas and closed areas, may have resulted in the subjects retreating into the darker, closed arms once an initial investigation had found no potential escape routes from the apparatus; such a response was not an option in the hole-board test. If a shelter were added to the hole-board apparatus, locomotion might be predicted to decrease over repeated exposure, with subjects choosing to remain under a shelter once potential escape routes had been investigated.
In support of previous reports (e.g. Aguilar et al., 2003; Ray and Hansen, 2004), our results indicate that female rats head-dip more frequently, locomote more and spend more time in the centre of the hole-board apparatus than males. These sex differences in behaviour were not affected by repeated exposure to the apparatus, or by the presence of novel objects. Given that the locomotor patterns of female rodents are influenced by the stage of the estrus cycle and by experimental manipulation of hormones such as estradiol (e.g. Garey et al., 2001; Morgan and Pfaff, 2002), researchers have questioned whether gonadal hormones might influence general levels of arousal that might impact on how females respond to novelty compared to males (Morgan et al., 2004). However, while reports of sex differences in hole-board performance have been interpreted as showing that females are more exploratory than males, females have been reported to locomote more than males in familiar, as well as novel, environments (e.g. Cortright et al., 1997; Eckel and Moore, 2004), and, as discussed above, head-dipping may not represent neophilic behaviour. Therefore, the conclusion that females are more exploratory or neophilic than males should not be drawn from such data.
The results of this study indicate that the assumption that head-dipping on the hole-board task represents a neophilic response is not necessarily accurate. At least on first exposure to the apparatus, head-dipping is likely to result from an attempt by the subject to find a potential escape routes. This issue highlights a more general problem with unconditioned behavioural tests of exploratory behaviour, in that high levels of locomotion around an environment should not immediately be interpreted as neophilia or a positively rewarded aspect of novelty-seeking (Welker, 1957). Any behaviour in a novel environment will be influenced by both neophilia and neophobia, such that a single behavioural measure is unlikely to be purely indicative of either neophilic or neophobic tendencies. Rather than being at polar ends of a continuum, neophilia and neophobia should be thought of as two orthogonal factors that can vary independently (Greenberg, 2003). For researchers interested in rodent exploration, simple measures, such as head-dipping, are unlikely to expose by themselves the complex interaction between these two factors.

exploration rat hole-board head-dipping neophilia neophobia