Voiding urosonography with ultrasound contrast agents for the diagnosis of vesicoureteric reflux in children

Voiding urosonography (VUS) encompasses examination of the urinary tract with intravesical administration of US contrast agent (UCA) for diagnosis of vesicoureteric reflux (VUR). The real breakthrough for US examination of VUR came with the availability of stabilized UCAs in the mid-1990s. This article presents a comprehensive review of various procedural aspects of VUS. Different US modalities are available for detecting the echogenic microbubbles: fundamental mode, colour Doppler US, harmonic imaging and dedicated contrast imaging with multiple display options. The reflux is graded (1 to 5) in a similar manner to the system used in voiding cystourethrography (VCUG). The most commonly used UCA for VUS, Levovist, is galactose-based and contains air-filled microbubbles. The recommended concentration is 300 mg/ml at a dose of 5–10%, or less than 5%, of the bladder filling volume when using fundamental or harmonic imaging modes, respectively. There are preliminary reports of VUS using a second-generation UCA, SonoVue. Here the UCA volume is less than 1% of the bladder filling volume. There is no specific contraindication to intravesical administration of UCA. The safety profile of intravesical Levovist is very high with no reports of side effects over a decade of use in VUS.
Introduction
Diagnostic imaging for vesicoureteric reflux (VUR) is a common procedure in children. Currently, three modalities are available for reflux diagnosis, namely voiding cystourethrography (VCUG), radionuclide cystography (RNC) and voiding urosonography (VUS). The last of these is carried out using US in combination with intravesical administration of US contrast agent (UCA). This allows the use of ionizing radiation to be avoided, which is not the case in RNC and VCUG. However, in VCUG a marked reduction in radiation dose has been achieved with the introduction of digital pulsed fluoroscopy.
The first attempts at the implementation of US for the diagnosis of VUR began in the mid-1970s. A comprehensive account of the evolution of this undertaking over the subsequent two decades has been presented by Darge [1]. The indirect methods for reflux diagnosis were based on US of the urinary tract, without administration of any kind of substance into the bladder. These included depicting various sonomorphological changes of the urinary tract as a result of VUR, detecting newly appearing or an increase in existing ureteral or pelvicalyceal dilatation during voiding and assessing ureteric jet changes with duplex and colour Doppler US. The direct means used to diagnose VUR required instilling different substances intravesically. The most frequently administered fluid was physiological saline solution. Ballooning of the renal pelvis during the filling of the bladder was the criterion for diagnosis of VUR. Application of air bubbles, by shaking the normal saline before administration or adding carbon dioxide, were also tried. US studies were also carried out, in which the empty bladder was solely filled with air. In addition to low diagnostic accuracy, all the above methods had major procedural drawbacks making them impractical for widespread integration into routine imaging.
The intravesical use of a UCA consisting of sonicated albumin (Albunex; Molecular Biosystems, San Diego, Calif.) for VUS in a child was first reported in 1994 [2]. Another UCA used in the past was Echovist (Schering, Berlin, Germany), which is composed of galactose with incorporated microbubbles [3]. Its very short imaging window of approximately 5 min, however, prevented its routine application. The breakthrough in US diagnosis of VUR in children came about the mid-1990s with the availability of UCAs containing stabilized microbubbles. Levovist (Levograf, Schering Spain, Madrid, Spain; SHU-508-A, Schering, Berlin, Germany) was the first such UCA to become available for clinical use in Europe. This opened the door for rapid development of VUS and its introduction as part of the routine diagnostic imaging option of VUR.
A number of different names and acronyms have been put forward to denote US examination for the diagnosis of VUR using intravesical UCAs. These include simply “sonography/ultrasound” [4–6], “reflux sonography” [7], “cystography” [8–10], “cystosonography” [11–15], “cystourethrosonography” [16] and “urosonography” [17–23]. They are more often used in combination with one or more of the following terms: “echo-enhanced”, “contrast-enhanced” and “voiding”. Depending on the US technique employed further descriptions such as “colour Doppler US” or “harmonic imaging” are added. The most widely applied name “voiding urosonography” with the abbreviation “VUS” was proposed for the first time in 2000 [24]. The selection of this particular designation was based on careful consideration of various factors. The prefix “uro-” is used to denote the bladder, ureters and kidneys. “Sonography” was chosen rather than “ultrasonography” as it is a shorter form. Thus “urosonography” correctly describes the fact that in this examination US of the bladder, ureters and kidneys takes place independent of the absence or presence of VUR. “Voiding” was selected instead of “micturating” as it is used more commonly in the medical literature. Despite the fact that for VCUG x-ray contrast agent is necessary, this is not directly added to the name VCUG in the form of, for example, contrast-VCUG. Likewise, the terms “contrast” or “echo-enhanced” were not added by default to the basic term “voiding urosonography”. The abbreviation “VUS” is not only similar to the most common acronym used for the radiological reflux examination, namely VCUG, but had also not been used in the medical literature to denote something else [25]. For the sake of minimizing the confusion of names and facilitating communication, literature search and procedural standardization, the use of one name for the same procedure, “voiding urosonography” (“VUS”) is recommended.
This article is part I of a comprehensive review of all currently available literature on VUS and prepares the ground for objective evaluation and decision-making. In this part (part I) a detailed procedural description is presented, including discussion of the pros and cons of the various examination steps and imaging modalities for VUS. The measures undertaken to optimize VUS are elaborated. Furthermore, studies dealing with adverse events of intravesical administration of UCAs are reviewed. In part II a review of comparative studies between VUS and RNC/VCUG is presented [26].
US contrast agent
The most widely used UCA for VUS is Levovist. This first-generation UCA was introduced for intravenous (IV) use in the mid-1990s and from 1999 started to obtain approval for intravesical application in children successively in 13 European countries and Australia. Levovist consists of dry granules made of galactose and palmitic acid [27]. The granules easily disintegrate into microparticles upon preparation. They form porous structures, which are necessary for the formation of bubbles. Gas bubbles are formed within the pores during suspension of the granules, while dissolution of the galactose particles begins simultaneously. The Levovist bubble is a microbubble of air—65% nitrogen and 35% oxygen—stabilized by palmitic acid. One gram of Levovist granules contains 999 mg d-galactose and 1 mg palmitic acid. The concentrations of microbubbles in freshly prepared batches (300 mg/ml) are within the range of approximately 1–2×108 microbubbles per millilitre of suspension. The Levovist suspension should always be freshly prepared prior to intravesical administration. The steps of preparation, as outlined by the manufacturer, must be strictly adhered to in order to avoid any reduction of contrast enhancement due to improper handling [28]. The administration of freshly prepared UCA has to be carried out within 30 min [29].
Concentration
The recommended concentration of Levovist for VUS is 300 mg/ml [28]. This concentration has been used for VUS in most studies. A concentration of 200 mg/ml seems to result in faster dissipation of microbubbles, as found in in-vitro experiments (unpublished data). In only three studies has a concentration of 200 mg/ml been used [13, 15, 30]. Bosio initially used this concentration, but later changed to the higher one as the conspicuity of refluxing microbubbles in fundamental mode was inadequate [13]. In another study in adults with transplanted kidneys this concentration in combination with an increased volume, i.e. 10–15% of bladder filling, was used [30]. The combination of colour Doppler US with the burst technique administering 200 mg/ml selectively in infants has been reported, but the results using just this concentration were not analyzed separately [15]. The utilization of the higher concentration of 400 mg/ml has only been reported once without any additional advantages being mentioned [31]. Probably, the disadvantage would be an increase in UCA volume leading to an unwarranted increase in cost.
Dosage
The volume of UCA administered has been rather variable. Some have applied a fixed dose for all, independent of the patient’s age, weight and bladder volume [4, 5]. In one study the volume was calculated in terms of body weight [13]. In most studies the volume of bladder filling is taken as the decisive factor [1, 12, 28]. This is very logical when considering intravesical administration of UCA and contrast enhancement of bladder content. It is possible to directly measure the bladder volume using the formula for an ellipse (length×width×height×0.5], note the total volume of normal saline administered in the bladder and also calculate in a simple way the maximum bladder capacity: volume in millilitres = (age in years +2)×30 [32]. Furthermore, using the bladder volume for calculation of UCA dose makes comparison between studies much easier.
Nakamura et al. [21] carried out simultaneously VCUG and VUS in 56 children. The fundamental (i.e. conventional) imaging modality was used for VUS. They initially administered Levovist into an almost empty bladder followed by continuous infusion of the radiographic contrast agent. They simultaneously monitored the appearance of reflux during VUS, relating it to the concentration of the UCA in the bladder. The concentration of UCA in the bladder at one point in time ranged form 1.8% to 23%. All false-negative results in the VUS were associated with a Levovist volume of <5% of the total bladder filling. Thus a volume of UCA between 5% and 10% of the bladder filling is required when performing VUS using fundamental imaging. As there is a clear correlation between the experience of the sonographer and the sensitivity of VUS, it is advisable for beginners to start with the highest recommended volume of 10% and with increasing experience to reduce the amount, but without going below 5% of the bladder filling when using fundamental imaging. It seems possible to reduce the UCA volume when adding colour Doppler US [15]. There is clear evidence that when using harmonic imaging, 5% or less of UCA with respect to the bladder filling will suffice [18, 33]. Preliminary experience with dedicated contrast imaging modalities such as cadence agent detection imaging (ADI) (Acuson; Siemens, Mountain View, Calif.) point to the potential of further dose reduction [34].
Physicochemical properties
UCAs were primarily developed for IV use and, consequently, preclinical studies were aimed at elucidating problems that could potentially have been encountered during administration via this route. Intravesical use is different and has its own peculiarities one must be aware of. An in-vitro study was carried out with Levovist to measure how variations in US machine setting, transducer choice, mode of application and bladder content may affect the microbubbles [35]. The effects of power output, transducer frequency and injection rate were found to be comparable to those during IV administration. The physicochemical properties of particular relevance for intravesical use are described below.
Interaction with normal saline solution
Unlike in VCUG and direct RNC, normal saline plays an important role in VUS. It is the actual bladder filling medium in which the microbubbles are injected. In an in-vitro study the median contrast duration when the UCA was mixed with normal saline solution from glass containers was 30 s, whereas when the same UCA was added to normal saline solution from a plastic container the median contrast duration increased to 11 min [36]. None of the plastic containers were sealed under vacuum. In contrast, most of the glass containers were sealed under vacuum. The mean pO2 of the normal saline solutions from vacuum-sealed containers was found to be 50% less than the pO2 of the normal saline solutions from plastic containers. The air-filled microbubbles of Levovist are permeable to air, and gas is exchanged with the atmosphere until equilibrium is reached [27]. Thus, in normal saline solution with low air saturation the microbubbles tend to collapse promptly [37]. Collapse of the microbubbles means that echo enhancers are not available. In practice, the use of normal saline solution from plastic containers is preferred.
Interaction with urine
Contrast duration of the UCA has been found to be more than four times longer in urine than in normal saline. It has been shown that simply adding urea to buffer the solution significantly increases contrast duration [38]. It is postulated that urea affects the bonding between water molecules and facilitates the formation of more microbubbles. Thus, having some urine in the bladder when performing VUS actually has a positive effect on imaging.
Ascent of microbubbles in the ureter
Once reflux of microbubbles takes place from the bladder into the distal ureter, the question as to whether the microbubbles can ascend passively to the proximal ureter and renal pelvis while the refluxed fluid stays in the distal ureter has been raised [39]. Given constancy of gravity, the density difference between gas and fluid and the viscosity of fluid, the velocity of ascent is proportional to the square of the microbubble radius. The typical microbubble radius of Levovist is 1–2.5 μm. Calculation of velocity of ascent indicates that for all practical purposes passive ascent can be excluded. Furthermore, in an in-vitro simulation of VUS with a set-up imitating the worst-case scenario (90°, glass tube (no adsorption), constant stirring of fluid suspension, and absence of counter-flow) the possibility of passive ascent was evaluated using an ultraviolet spectrometer for the detection of microbubbles. The result of this experiment was also negative. These findings suggest that the microbubbles in the ureter do not ascend passively and that reflux pressure is necessary for propagation, particularly in vivo with a constant counter-flow of urine from the renal pelvis to the bladder.
Procedural details
VUS entails four major basic steps: (1) scan of the urinary tract before contrast agent administration, (2) intravesical administration of prewarmed physiological saline solution and UCA, (3) scan of the urinary tract after administration of UCA, and (4) scan of the urinary tract after administration of UCA during and after voiding [28, 40]. Optionally, a transperineal US of the urethra and/or cyclical filling of the bladder may be added. Various US modalities are available for performing VUS. The main differences between the modalities are in the degree of conspicuity of the microbubbles and sensitivity of reflux detection.
US scan modalities
Fundamental imaging
This conventional modality in B-mode is the earliest and most widespread modality employed for VUS. In the case of fundamental imaging it is important to use the same scan planes and magnification of the ureters and renal pelves before and after UCA administration in order to facilitate comparison of the images (Fig. 1). This is particularly important when the reflux is not obvious.
Fig. 1Scans in fundamental mode before (a, c) and after (b, d) contrast agent administration of a dilated left distal ureter (a, barrow) and pelvicalyceal system (c, d). In the postcontrast scans echogenic microbubbles fill the distal ureter (b) and are also detected in the pelvicalyceal system (d)
Colour Doppler US
The microbubbles increase the backscattered signal from the urine giving it a characteristic chromatic effect, e.g. a set of blue and red punctiform colour collection [32]. This makes recognition of the urine flow direction easier and enhances the visualization of the microbubbles in the pelvicalyceal system. It is recommended that the Doppler US settings be optimized to perform VUS [31, 41]. Currently, there are five reports in which colour Doppler US alone or in combination with fundamental imaging have been used for VUS [11, 14, 31, 41, 42]. The diagnostic accuracy of colour Doppler US was compared with fundamental imaging in only one study [41]. In the same group of patients VUS without and with colour Doppler US followed by VCUG was carried out. With VCUG as the reference method, the diagnostic accuracy was found to be 96% with colour Doppler US and 90% without. In another study, no significant increase was found in the sensitivity and specificity of VUS when adding colour Doppler US [42]. In one study a modified colour Doppler US examination was used employing a high mechanical index (MI) burst contrast technique based on stimulated acoustic emission (SAE), in which the microbubbles are made to burst creating strong acoustic signals [15]. It was possible to detect more reflux with the burst colour Doppler US compared to regular colour Doppler US or fundamental imaging and even VCUG. No systematic evaluation is available regarding the application of power Doppler US for VUS. Overall, adding optimized colour Doppler US to fundamental imaging seems to have some advantage and should be tried when other contrast-specific modalities are not available.
Harmonic imaging
The nonlinear propagation property of US waves is the basis of harmonic imaging [43]. This may be optimized to receive harmonics from either tissue or microbubbles, thus creating “tissue-specific harmonic imaging” or “contrast-specific harmonic imaging”, respectively. Some US machines only use the second harmonic for imaging (narrow band), whereas others are capable of implementing a wider range of harmonics (wide band). There may be some degree of difference in image quality between these different technical approaches, in general the latter being of higher quality.
Harmonic imaging increases contrast and spatial resolution and also results in a reduction of artefacts [43]. The resulting images are clearer and crisper. The advantage of this modality compared to fundamental imaging for scanning the urinary tract in children has already been shown [44]. Due to attenuation, at a certain depth the positive effect of harmonic imaging will disappear. Regarding contrast-enhanced imaging, there is distinctly increased conspicuity of the microbubbles with harmonic imaging (Fig. 2). Air-filled bowel can be a disturbing factor, particularly when imaging the retrovesical space, requiring adjustment of the gain. Darge et al. [17] compared fundamental and harmonic imaging options in 54 children undergoing VUS. In all cases the conspicuity of the microbubbles was much higher with harmonic imaging than with the fundamental modality. Moreover, there was an increase in the reflux detection rate of 30% (from 19 to 27 pelvic-ureter units, PUUs). In this study the volume of Levovist used was 10% with respect to bladder filling. Even reducing the volume of UCA, more reflux episodes were detected in VUS with harmonic imaging than in VCUG [18, 33]. If available, harmonic imaging should be chosen in preference to the fundamental modality for VUS [17, 18, 33].
Fig. 2Scans after contrast agent administration in fundamental mode (a, c) and with harmonic imaging (b, d) of the bladder and right dilated ureter (a, bdotted circle) and a duplex kidney (c, d) with a multicystic dysplastic upper moiety. Reflux in the right ureter and in the lower moiety of the duplex kidney (grade II, arrow) are much more conspicuous with harmonic imaging (b, d). Note also the crisper depiction of the cysts in the upper moiety with harmonic imaging
Dedicated high-MI contrast imaging with multiple display options
In recent years, there have been major developments in US technologies for contrast-enhanced US. One such US modality uses high-MI imaging resulting in destruction of microbubbles and depicts the bubble destruction signature as a colour overlay with the possibility of visualizing the grey-scale image alone, the grey-scale image together with the microbubbles with colour overlay or just the microbubbles with colour overlay alone. Additional features may be real-time dual imaging in which two of the above modalities can be used in parallel and the possibility to switch between the three different presentations after having acquired an image in just one option (Figs. 3 and 4). This modality is named differently by different manufacturers: for example, agent detection imaging (ADI; Sequoia, Acuson Siemens), contrast tissue enhancement imaging (CTEI; Technos MPX, Esaote), rate subtraction imaging (RSI; Aplio, Toshiba), true agent detection (TAD; Logiq 9, GE), etc. [45]. The increase in microbubble conspicuity with this modality is so striking that even a beginner should find the examination easy to perform. The potential for further reduction of the dose of UCA and duration of examination are enormous and require future evaluation. If available, this US technique is preferred for VUS using Levovist. This feature is also available with low-MI imaging, which is important when using newer generation UCAs.
Fig. 3VUS with the application of dedicated contrast imaging modality using high-MI (agent detection imaging, ADI). Reflux into the right ureter (arrow) and right renal pelvis (grade II). a Using grey-scale display alone the refluxing microbubbles are not easily demonstrated but the renal parenchyma is seen well. b Using “grey scale + contrast” visualization of both the reflux with a colour overlay and the bladder and kidney are noticeably improved. c Using “contrast only” the tissue part has been subtracted and only the microbubbles are seenFig. 4VUS with the use of a dedicated contrast imaging modality using high-MI (agent detection imaging, ADI). Note the time and number of the images (arrow). Once just one image has been documented it is possible as a postprocessing option to switch between the different modalities for display: a “grey-scale + contrast” and b “contrast only” options. In this case of grade III reflux marked intrarenal reflux is present in the upper pole (arrowhead)
Procedural steps
Precontrast scans of the bladder, ureters and kidneys
The necessity to perform a detailed scan of the urinary tract arises when the VUS is combined with a follow-up US of the urinary tract, for example after pyelonephritis or when there is a need for comparison with the postcontrast images. The scan before contrast agent administration should be carried out in accordance with standard US of the urinary tract in the supine position (and/or the prone position), in both the transverse and longitudinal planes [28]. Particular attention should to be paid to documentation of subtle changes in the retrovesical region, the vesicoureteric junction and any dilated ureter. The renal pelves (and/or the calyces) are imaged with maximum magnification. A scan in the supine position may suffice if adequate visualization of the renal pelves on both sides is possible, even though scanning the kidneys with the child in the prone position definitely allows better demonstration of the pelvicalyceal system. It should always be kept in mind that not performing a precontrast scan reduces the duration of the whole examination. This should be the case whenever harmonic or other dedicated contrast imaging is used.
Intravesical administration of normal saline and US contrast agent
The UCA and normal saline are administered via a transurethral catheter, but suprapubic puncture is also possible. The administration of UCA is carried out under US monitoring. The aim should be to have homogeneous strong contrast agent density of the bladder contents. Early studies advocated filling the bladder to the maximum prior to injecting the UCA [13, 28]. This makes the calculation of UCA volume to be administered easier, but has the disadvantage that low-pressure reflux may be obscured. Moreover, in neonates and infants due to repeated voiding at small bladder filling volume, there is insufficient time to administer UCA and scan the urinary tract. Increasingly, a more fractional approach to administering UCA is being practised [21, 40]. It is important to note that emptying the bladder for the sake of UCA administration is not mandatory as explained above. It is helpful to inject UCA into a bladder that is not completely empty in order to have better visualization of the microbubbles and avoid a strong dorsal acoustic shadow. Using a three-way valve it is easy to alternately administer normal saline and UCA adjusting the volume of UCA to the bladder filling volume and in the end reaching the recommended dose. The Levovist suspension should be administered slowly for two reasons: first, to minimize the destruction of the microbubbles and second, to reduce settling of the suspension at the bottom of the bladder that would create an acoustic shadow that temporarily obscures the retrovesical region [28]. In such a case, turning the child several times from side to side hastens the homogeneous distribution of the UCA in the bladder. The administration of normal saline is continued until the child has the urge to micturate or there is the first slight sign of back pressure to the infusion or injection.
Postcontrast scan of the bladder, ureters and kidneys
The US scan after contrast agent administration is basically carried out in the same manner as that before contrast agent administration. There are minor procedural variations depending on the type of US modality utilized. The diagnosis of reflux is made when echogenic microbubbles appear in a ureter or renal pelvis. When a ureter is distinctly visible behind the bladder, the microbubbles render the echo-free lumen echogenic. When the ureter is not clearly visible as a round echo-free structure, VUR can be demonstrated if microbubbles are seen entering the vesicoureteric junction or if just behind this junction, one can depict a round echogenic spot in transverse section, which can be differentiated from its surroundings by its strong echogenicity and possibly constant movement of microbubbles [28]. When the reflux reaches the kidney the echogenic microbubbles can be detected in the pelvicalyceal system. In a recent study by Kopac et al. [23] the need for UCA to demonstrate reflux was again emphasized. They compared indirect non-contrast VUS with contrast-enhanced VUS in 47 children with 93 PUUs. In the former there was no catheterization and bladder filling and any increase in renal pelvic and proximal ureteric width during voiding was considered as a sign of VUR. Compared to VUS with UCA this was found to have low diagnostic accuracy of only 59%, which is not sufficiently reliable for routine use.
Postcontrast scan of the bladder, ureters and kidneys during and after voiding
The US examination is continued during and after voiding in a similar manner as above, always scanning the right and left kidneys alternately, and also the bladder if the patient’s position allows. If cyclic filling is not planned, the catheter can be removed prior to micturition. Most children can void around a thin catheter on the examination table while lying supine, prone or on the side [28]. Other alternatives to consider are to have the child sit on a potty and to scan the kidneys from the back. A “music potty” that signals voiding with music is helpful. In older boys the option to micturate into a urine bottle while standing and being scanned from the back may be offered. At the end the bladder is checked for residual urine.
Postcontrast scan of the urethra during voiding (urethrosonography)
The main focus in VUS has been on detection of reflux. In the past, transperineal imaging of the urethra was not given as much consideration during VUS. The lack of urethral imaging in VUS was regarded as a drawback compared to VCUG [46, 47]. In recent years there has been an increased interest in the inclusion of urethral imaging. To date, six studies are available that deal exclusively or partly with contrast-enhanced voiding US of the urethra [16, 19, 48–51]. In these studies the urethra was examined in a total of 847 children comprising 647 (76%) boys and 200 (24%) girls (age range 1 day to 15 years). Levovist in combination with fundamental imaging was used in all children. In half of the studies, both boys and girls were included and in the remainder only boys [19, 48, 49]. An unbiased comparison of all cases with VCUG was carried out in only two studies [48, 49]. In three studies just those children with pathological findings of the urethra by voiding urethrosonography underwent VCUG [19, 50, 51]. In the remaining one study the selection for comparison was not clearly presented [16]. The diagnosis of posterior urethral valve was made in 22 boys (3.3%) using transperineal contrast-enhanced voiding urethrosonography (Fig. 5). This was also found to be suitable for assessment of the urethra after valve resection [48, 49]. Other diagnoses in boys were anterior urethral valve and urethral stenosis [48]. All diagnoses were confirmed by VCUG. However, the urethral pathologies detected were not only too few, but also the range of pathologies encountered was small [47]. Other urethral pathologies such as paraurethral cysts, diverticula, double urethra, urethral fistula, and complex anorectal and cloacal malformations are still missing in these series. Urethrovaginal reflux was evaluated in two studies and was found to be present in 67 of 165 girls (40.6%) [19, 49].
Fig. 5Transperineal voiding urethrosonography (a) as part of VUS in comparison with (b) VCUG. To facilitate the comparison the US image (a) is presented upside down. Note in the transperineal US (a) the microbubbles in the bladder (B) and in the massively dilated posterior urethra (pU). The anterior urethra (aU) is depicted as very thin in the presence of a posterior urethral valve (arrow). The finding was confirmed on VCUG (b) (courtesy of Dr. M. Bosio, Milan, Italy)
The most important study comparing contrast-enhanced voiding urethrosonography and VCUG is that by Berrocal et al. [49]. In this prospective study a total of 146 children (87 boys, 59 girls) with a mean age of 3.3 years (8 days to 14 years) were recruited. In the voiding part of VUS, primarily transperineal US of the urethra was performed. During voiding the catheter was removed. The diameters of the anterior and posterior parts of the urethra were measured at maximum dilatation during voiding. In boys the normal value was found to be 6.1 ± 0.8 mm (2.8–7.1 mm) and 6.3 ± 0.67 mm (3.7–7.2 mm), respectively. In girls the urethral diameter was 4.2 ± 1.0 mm (2.5–7.8 mm). This was the basis for evaluation of pathological changes in the urethra. In all children the US study was followed by VCUG. There were three boys with posterior urethral valves detected on US with dilated posterior urethra, reduced diameter of the anterior urethra and delay in UCA flow at the valve. Furthermore, urethral stenosis was diagnosed in two boys. Seven children were evaluated after resection of the posterior urethra valves and were found to have a normal posterior urethral diameter in the presence of dilatation of the anterior part without any flow delay. In all children these findings were confirmed on VCUG. All girls and 75 boys showed a normal urethra at both transperineal US and VCUG. Thus sensitivity and specificity were each 100%. The approximate mean duration for VUS including transperineal voiding US was 30 min. In conclusion, transperineal contrast-enhanced VUS of the urethra has not only been shown to be possible but also to be a potential adjunct to VUS in routine examinations.
Cyclical filling of the bladder
VUR is an intermittent phenomenon. Differences in presence and degree of reflux on repeated examinations have been reported in both VCUG and RNC [52, 53]. In general, cyclical filling results in an increase in reflux detection rate. Repeated filling of the bladder may be necessary when there is marked discrepancy in kidney size and/or intermittent dilatation of the ureters on US and a negative result during the initial examination. VUS has a specific advantage in this regard as it is not associated with repeated exposure to radiation. Three studies have evaluated the value of cyclical filling in VUS [54–56]. In two of the studies, two cycles of bladder filling including Levovist administration were carried out with scanning in fundamental mode [54, 55]. One study was performed in 27 patients with transplanted kidneys [54]. This group comprised eight children (age range 12.2–18.3 years) and 19 adults (age range 19.2–54.8 years). The reflux detection rate in the first and second cycles was 17 and 16 of 27, respectively, and thus not very different, but there were significantly higher reflux grades in the second cycle. So the conclusion was that cyclical filling in VUS with fundamental imaging did not result in increased reflux detection rate. In another study with a greater number and wider spectrum of patients, exclusively children, the opposite was found. Novljan et al. [55] evaluated cyclical filling in 49 children (age range 1.4–15.8 years, mean 4.1 years) with 98 PUUs. A total of 35 PUUs with reflux were detected when taking both cycles into account. In the first cycle 28 of 35 and in the second 33 of 35 of the PUUs were positive. In the first cycle only just 2 of 35 refluxes (both grade II) were detected. The VUR diagnosed in the second cycle alone comprised six PUUs with grade II and one PUU with grade III reflux. Not only were 25% more refluxing units detected in the second cycle but also 50% more grade III refluxes.
When the bladder is completely emptied one can often still find residual echogenic UCA lining the bladder mucosa which together with the hypoechoic wall resembles the mouth part of a “smile sign” (Fig. 6). This so-called “smile sign” indicates that there is still enough UCA in the bladder to facilitate repeat filling with normal saline alone and allow another cycle of VUS. Papadopoulou et al. [56] found that in 112 of 117 children (96%) undergoing cyclical harmonic VUS the residual UCA in the bladder was sufficient to allow a second filling with normal saline only. In the 112 children (224 PUUs) they compared the results of the first cycle with Levovist and normal saline administration with those of a second cycle with administration of only normal saline. VUR was detected in 57 PUUs in the first cycle, and of the remaining 167 nonrefluxing PUUs, 12 showed VUR in the second cycle (one grade I, nine grade II, two grade III). Only one PUU reflux (grade II) was diagnosed in the first cycle but missed in the second cycle. Considering all refluxing units as true positives, the sensitivities of the first and second cycles were 87% and 98% and the negative predictive values 84% and 98%, respectively. Thus a second cycle of harmonic VUS with no addition of UCA reveals significantly more PUUs with VUR at almost no additional cost for the examination. From the current standpoint of available data, cyclical filling in VUS, particularly when using harmonic imaging, seems to be a very promising adjunct.
Fig. 6At the end of VUS and bladder emptying there can still be a “layer” of UCA lining the mucosa of the empty bladder. The configuration of the bladder in transverse section in combination with the remaining echogenic UCA resembles the mouth part of a “smile sign”. This sign indicates that there is still sufficient UCA in the bladder and if considered necessary a second filling with normal saline only would suffice to carry out cyclical VUS
Grading of reflux
VUR grading is necessary because the severity of reflux correlates with prognosis and consequently serves as a basis for therapeutic decision making. It is also essential to have a uniform grading system for comparison between different research studies. The reflux gradings are based on the degree of pelvicalyceal and ureteral dilatation. Intrarenal reflux is not taken into consideration for grading purposes. It is to be noted that it is possible to depict intrarenal reflux in VUS, particularly when using harmonic imaging and other high-MI modalities [13, 57]. No systematic evaluation of intrarenal reflux in VUS is available. The first grading system for VUS was proposed in 1985 by Beyer et al. [58]. Here grading was for VUS with administration of normal saline only into the bladder. This sonographic grading has five levels based on the extent of dilatation of the renal pelvis, calyces and ureters. Taking this grading system and in conformity with the international reflux grading for VCUG, a five-level grading system was also adapted for contrast-enhanced VUS [59, 60]. The diagnosis of reflux is based on the presence of microbubbles and the severity of the reflux determined by taking primarily the pelvicalyceal and secondarily the ureteric dilatation into account (Table 1). This grading system for VUS has gained widespread acceptance. With the severity of reflux being a continuum, no sonographic measurements have been proposed for differentiating between the grades. Unavoidably, as in VCUG, there is a certain element of subjectivity in the grading system and also some reflux episodes do not fall precisely within one of the five grades. The attempt to differentiate between reflux in dilated and nondilated systems has not become widely used, probably due to lack of immediate consequence of such an addition [60].
Table 1Reflux grading in contrast-enhanced VUSGradeDefinitionIMicrobubbles only in the ureterIIMicrobubbles in the renal pelvis; no significant renal pelvic dilatationIIIMicrobubbles in the renal pelvis + significant renal pelvic dilatation + moderate calyceal dilatationIVMicrobubbles in the renal pelvis + significant renal pelvic dilatation + significant calyceal dilatationVMicrobubbles in the renal pelvis + significant renal pelvic dilatation and calyceal dilatation + loss of renal pelvis contour + dilated tortuous ureters
Duration of VUS examination
In four comparative studies, in which VUS and VCUG were carried out successively, the duration of each procedural step was recorded [12, 28, 31, 42]. In two of the studies VUS was carried out using fundamental mode [12, 28]. The other two studies incorporated colour Doppler US [31, 42]. In these studies the durations of VUS, including catheterization, were in the range 26–34 min with the precontrast US taking up almost one-third of the time. The durations of VCUG, adding the time for catheterization, were in the range 13–20 min. In one other study VUS and VCUG were carried out simultaneously and here the duration of one such examination including catheterization was 9 min [61]. Novljan et al. [55] performed VUS with cyclical filling (two fillings) and the mean duration of the VUS was 24 min (range 15–30 min). Overall, these studies show that the duration of VUS shows a wide range and is longer than that of VCUG. As mentioned above the precontrast US may not be necessary when using harmonic or other dedicated contrast imaging modalities, resulting in a reduction of the duration of VUS by almost one-third. This would put the duration of VUS equivalent to that of VCUG.
VUS with a second-generation US contrast agent (SonoVue)
In 2001 a second-generation UCA—SonoVue (Bracco, Milan, Italy)—was approved in the European Union for IV use in adults. SonoVue is now widely used, the main application being for the evaluation parenchymal abdominal lesions [62]. Even though it has not been approved yet for use in children one in-vitro and four clinical studies point out potential advantages compared to the use of the first-generation UCA, Levovist [63–67]. SonoVue is composed of a stabilized aqueous suspension of sulphur hexafluoride (SF6) microbubbles with a phospholipid shell and is available as a package comprising one vial with granules and a 5-ml syringe prefilled with normal saline [62]. In an in-vitro comparative study with Levovist (5% volume) the contrast duration with SonoVue was seven times longer at a dose that was 80% lower [63]. It was found that UCA at 0.25–1.0% of the filling volume would provide adequate contrast. Moreover, the contrast duration of a freshly prepared suspension of SonoVue was stable over 6 h, whereas Levovist showed a significant reduction after 30 min.
The first clinical studies [64–67], in total comprising 210 children, have shown that low-MI imaging is the most favourable US modality for VUS when using SonoVue [67] (Fig. 7). Although it is possible to use all other modalities with this UCA, the highest contrast difference between tissue and microbubbles seems to be achieved with low-MI imaging, in which the tissue is suppressed and the microbubbles become more conspicuous. During intravesical administration of SonoVue there is a minor difference compared with Levovist. With the patient in the supine position at the beginning of injection the UCA accumulates at the roof of the bladder and thus does not reach the vesicoureteric junctions [65]. Only after starting normal saline infusion can a homogeneous distribution of the microbubbles be observed. Not much acoustic shadowing of the retrovesical space is encountered. The intravesical dosages that have been successfully used in clinical studies are 1% of bladder filling [65] and 1 ml per bladder filling [67]. This implies that with a 5-ml suspension from one vial that is stable over 6 h there is the potential for performing several studies from the one vial. If one vial can be used for more than one patient, the cost of the UCA, a major obstacle to the widespread use of VUS, could be reduced. It is important to note that in some countries there may be restrictions regarding the use of one vial for more than one patient.
Fig. 7VUS using the second-generation UCA, SonoVue. The scan before contrast agent administration (a) uses the tissue harmonic imaging (THI) modality and the scan after contrast agent administration (b) uses echo contrast imaging (ECI). In the former the MI (arrow) is high (1.1) while in the latter a low-MI (0.2) technique is applied. In THI the kidney and particularly the renal pelvis are very well depicted. The refluxing microbubbles of the second-generation UCA are displayed most conspicuously using the low-MI imaging modality
Contraindications and adverse events from intravesical administration of US contrast agents
There are no specific contraindications as such to the intravesical administration of Levovist. There are five clinical studies including a total of 626 patients with an age range of 2 days to 20 years, in which systematic monitoring for possible adverse events related to intravesical administration of Levovist were carried out [12, 20, 28, 41, 42]. The evaluation incorporated all or some of the following: various levels of vital sign monitoring [12, 28], assessment for possible signs and symptoms during and after the procedure, observation for up to 12 h as inpatient [42], and request to the patient and parents to report any symptoms in the next 24 h and an active 24-h follow-up by phone. This evaluation was carried out within the context of comparative studies, in which VCUG was performed following VUS. All patients were catheterized for the examinations. There were 15 patients with transient visible haematuria during or at completion of voiding at either VUS or VCUG and one patient each with transient mild abdominal and urethral pain. For these observed adverse events the catheterization was primarily to blame rather than the UCA [68]. No substance-specific adverse events definitely related to the intravesical administration of Levovist were observed.
The osmolality of Levovist in solution is about five times higher than that of blood [37]. This osmolality corresponds to that of previously used ionic radiographic contrast agents, which were likewise instilled into the bladder. No adverse events attributable to the osmolality were observed with these agents. Apart from this, the amount of UCA administered is small and in the bladder there is usually urine and/or normal saline, resulting in dilution of the hyperosmolar solution. Hyperosmolality of the UCA can, therefore, be ignored in VUS, particularly as it lies approximately in the physiological range of osmolality of urine. No animal study has been published on the potential effect of insonation of the urothelium in the presence of microbubbles, particularly at high MI settings. Potential alterations of the urothelium cannot be excluded with absolute certainty. Over the last 10 years, predominantly in Europe, it is roughly estimated that some 20,000 VUS examinations have been conducted in children (publications, reports and personal communications). There have been no reports of clinical adverse effects related to the intravesical administration of Levovist.
Experience with the new UCA, SonoVue, for VUS is still very limited. Contraindications for the IV administration are known hypersensitivity to sulphur hexafluoride and certain cardiopulmonary disorders [29]. Recently, a large-scale retrospective analysis showed that IV SonoVue has a good safety profile in abdominal applications [69]. In the small number of VUS studies with a total of 210 children no adverse events directly related to the UCA have been reported [64–67]. There are, as yet, no detailed studies regarding any possible interaction with urine and the urothelium.
Conclusion
It is important to realize that a breakthrough to implement US for reflux diagnosis, which had began in the late 1970s, only came with the availability of a stable UCA. Advances in US technology resulting in marked improvement in the depiction of microbubbles have facilitated many procedural aspects in addition to the diagnostic accuracy of the modality. Furthermore, the first results of VUS with the use of a more stable second-generation UCA point to further procedural and diagnostic progress. Like VCUG and direct RNC, it is still necessary to catheterize the bladder or perform suprapubic puncture for VUS. This is a major drawback of all direct reflux examinations and one that makes them invasive. Attempts to generate bubbles exogenously using high-intensity focused US would solve this problem, but have not yet moved beyond the stage of animal studies [70].

voiding urosonography
ultrasound contrast agent
vesicoureteric reflux


Gray matter injury associated with periventricular leukomalacia in the premature infant

Neuroimaging studies indicate reduced volumes of certain gray matter regions in survivors of prematurity with periventricular leukomalacia (PVL). We hypothesized that subacute and/or chronic gray matter lesions are increased in incidence and severity in PVL cases compared to non-PVL cases at autopsy. Forty-one cases of premature infants were divided based on cerebral white matter histology: PVL (n = 17) with cerebral white matter gliosis and focal periventricular necrosis; diffuse white matter gliosis (DWMG) (n = 17) without necrosis; and “
Introduction
Periventricular leukomalacia (PVL), a major disorder of the immature cerebral white matter, has long been considered the underlying neuropathologic substrate of cerebral palsy in premature infants who survive into childhood [41]. The substrate of the cognitive impairments in these children, however, is less certain, given that cognition is typically attributed to gray matter (neuronal cell body), as opposed to white matter (oligodendrocyte), function, and the incidence of gray matter injury in PVL has historically been considered minimal [1, 4, 8]. Understanding the neuroanatomic basis of impaired cognition is of increasing importance in the care of premature infants: currently, in extremely premature infants (born at <1,500 g), the most common neurological disabilities involve cognition and learning, affecting 20–50% of such infants, compared to cerebral palsy which affects 10% [39, 42]. Attempts have been made to correlate neuropathologic findings in autopsied brains with neurologic sequelae in survivors of premature birth, however, much of this work has focused on the role of white matter injury and hemorrhages and not gray matter injury [13]. In the seminal paper of the neuropathology of PVL in 1969, Banker and Larroche reported only “mild neuronal injury” in the cerebral cortex, hippocampus, subiculum, basis pontis, and cerebellar dentate nucleus and Purkinje cells in the majority of PVL brains [4]. Subsequent neuropathologic studies reported only rare “anoxic neuronal injury” in association with PVL [1, 8], or downplayed the importance of observed gray matter lesions and did not emphasize their potential importance to neurologic sequelae in survivors. Thus, the conventional teaching has been that cerebral white matter is especially vulnerable to hypoxia-ischemia in the premature brain, with relative sparing of the gray matter, and that gray matter injury dominates only in older infants, children, and adults [23, 24], even though recent evidence shows that neuronal/axonal injury is common in the perinatal brain [5, 25]. This teaching is increasingly challenged by modern quantitative volumetric MRI studies of premature infants who exhibit reduced volumes of the cerebral cortex [35], thalamus [17, 18], basal ganglia [17, 18], and hippocampus [19, 34]. The anatomic substrate for the gray matter volumetric deficits associated with the apparent white matter disease remains unknown.
In the following study of the neuropathology of premature infants autopsied at this institution in the modern era of neonatal intensive care, we aimed to determine: (1) whether gray matter abnormalities in premature infants are more common in the presence of PVL than in the absence of this lesion; and (2) whether the constellation of any gray matter abnormalities provides insight into the basis of the cognitive impairments in living premature infants. In this study, we stratified the cases according to three patterns of cerebral white matter histology: (1) PVL (n = 17), defined as diffuse cerebral white matter gliosis combined with focal (macro- and/or microscopic) periventricular necrosis and; (2) diffuse white matter gliosis (DWMG) (n = 17), defined as diffuse gliosis without focal necrosis; and (3) “Negative” (n = 7), without diffuse gliosis and focal necrosis. While the pathogenesis of the white matter gliosis in the DWMG group is unknown, we considered it in a separate category from PVL (gliosis combined with focal necrosis) because the cerebral white matter pathology is distinct, without macro- and/or microscopic periventricular cysts. In the following study, we analyzed the three groups independently of each other. We hypothesized that PVL cases have a significantly greater incidence and degree of gray matter injury than non-PVL, i.e., DWMG and Negative cases; that this injury involves structures critical for cognition and learning, i.e., deep gray nuclei, cerebral cortex, and hippocampus; and that the pattern of gray matter injury mimics the pattern of volume reduction in the deep gray nuclei and cerebral cortex detected by neuroimaging studies in long-term survivors.
Materials and methods
Case selection criteria
The neuropathology of all premature infants (<37 gestational weeks at birth) autopsied between 1997–1999 at Children’s Hospital Boston was retrospectively reviewed. This time-frame was selected because it represents a modern era of intensive care management of premature infants in a Level 3 neonatal intensive care nursery. It also represents a period at our hospital when the brain and spinal cord were extensively sampled in a relatively standardized fashion, whether or not macroscopic lesions were apparent, thereby permitting a systematic neuropathologic survey in a large dataset. Parental authorization of the use of autopsy human tissue for research was given in each case.
Microscopic slide review
A median of 15 (range 9–20) hematoxylin–eosin (H&E) or H&E/Luxol-fast-blue stained sections was examined from each case. These sections included cerebral cortex from all lobes, thalamus (at the level of lateral geniculate nucleus and including the dorsomedial and lateral posterior nuclei), hypothalamus, caudate, putamen, globus pallidus, hippocampus (level of lateral geniculate nucleus), amygdala, cerebellar dentate nucleus, cerebellar cortex (including Purkinje cells and granule cell layer), midbrain, pons, and medulla.
We scored the density of neuronal necrosis, neuronal loss, and gliosis in the most severely affected high power fields of each of the gray matter sites. Two of three neuropathologists (CRP, RDF, HCK) reviewed the slides together at any one time, with a three-way consensus achieved on difficult cases. The brainstem tegmentum was graded separately from the basis pontis and inferior olive in recognition of their susceptibility to injury in the perinatal period [23]. We defined neuronal necrosis as hypereosinophilic neurons with pyknotic nuclei, or, in cases with neuronal immaturity and scant cytoplasm, karyorrhexis [2]. We used the following scale: 0, no necrosis; 1, a few scattered necrotic neurons in a high-powered field (hpf; 400×); 2, isolated clusters of necrotic neurons/hpf; and 3, larger, confluent areas of necrotic neurons/hpf. We scored neuronal loss as: 0, no neuronal loss/hpf; 1, mild, scattered neuronal dropout/hpf; 2, moderate, focal areas of neuronal dropout/hpf; and 3, severe, confluent areas of neuronal dropout/hpf. We interpreted neuronal necrosis as an acute or agonal change, occurring within 24–48 h of death [2], while neuronal loss and gliosis were interpreted as markers of subacute or chronic injury, indicative of insult occurring 3–5 days or more prior to death [2]. We combined cases for analysis that had grades 2/3 and 3/3 of neuronal loss because these grades are unequivocally recognized by standard microscopic examination, and therefore represent, in our opinion, a substantial degree of injury. We scored gliosis as: 0, no reactive astrocytes/hpf; 1, 1–10 reactive astrocytes/hpf; 2, 11–20 reactive astrocytes/hpf; and 3, >20 reactive astrocytes/hpf. Reactive astrocytes were defined as stellate configured cells with abundant (“hypertrophic”) eosinophilic cytoplasm and an enlarged, often eccentrically placed nucleus with delicate chromatin. The cytoplasm of these cells was immunopositive for glial fibrillary acidic protein (GFAP), the well-established astrocytic marker. In scoring neuronal necrosis, neuronal loss and gliosis, we evaluated the entire available region of each gray matter structure in the section, and assessed the most severely injured region, which was virtually always representative of all fields.
We assessed white matter in the cerebral lobes, corpus callosum, posterior limb of the internal capsule, and cerebellum for PVL and diffuse white matter gliosis (DWMG). PVL is defined by the combined presence of: (1) focal necrosis in the periventricular region; and (2) diffuse reactive gliosis in the surrounding white matter [24]. DWMG is defined by the presence of gliosis in the cerebral white matter unaccompanied by periventricular foci of necrosis [24]. Both PVL and DWMG are characterized by reactive astrocytes throughout the white matter, and are thus associated with a pattern of “diffuse” white matter injury. The density of white matter gliosis was scored according to the same scale used for gray matter gliosis (grades 0–3).
Glial fibrillary acidic protein (GFAP) immunohistochemistry
Four-micron thick formalin-fixed paraffin-embedded sections of frontal cortex could be cut from 27 of the 41 cases and were immunostained with mouse anti-GFAP antibody (1:500, #SM1-22R, Covance, Berkeley, CA). Negative controls were performed without primary antibody. Scoring of GFAP stained sections was performed by counting positive cells/hpf, in the most intensely immunopositive region of frontal cortex after a survey of all fields. Reactive astrocytes were defined as those cells with substantial cytoplasmic GFAP staining around a nucleus. Other non-reactive cortical astrocytes with limited cytoplasmic staining had GFAP positive processes that were generally perpendicular to the glial limitans were counted separately. The grading system was: 0, no staining; 1, 1–10 cells/hpf; 2, 11–20 cells/hpf; and 3 > 20 cells/hpf. Two observers (CRP, HCK) scored each case without knowledge of the white matter group.
O4 and GFAP double-labeling immunofluorescence
Myelination gliosis is commonly encountered in the newborn brain and must be discerned from reactive gliosis. So-called myelination glia are oligodendrocyte precursors that form during myelination; these cells have large nuclei with chromatin intermediate in density between oligodendrocytes and astrocytes and large slightly basophilic cell bodies so they can potentially be mistaken for reactive astrocytes [36]. To help discern reactive gliosis from myelination gliosis fresh tissue that was immediately fixed in 4% paraformaldehyde and sectioned at 40–50 μm was available from 4 PVL, 1 DWMG and 4 of the Negative cases. Double labeling was performed sequentially beginning with the mouse anti-O4 monoclonal antibody (1:750; gift from Dr. Steven Pfeiffer) to detect developing oligodendrocytes and followed with rabbit anti-GFAP antibodies (1:200, Z0334, Dako) to detect astrocytic differentiation. Relevant secondary antibodies conjugated with FITC or Texas Red were used and sections were visualized with Nikon Eclipse E800 microscope (Nikon, Melville, NY) outfitted with Spot image capture software (Diagnostics Instruments Incorporated, Sterling Heights, MI).
Statistical analysis
The 41 cases were divided into three groups according to cerebral white matter histology: (1) a PVL group; (2) a DWMG group; and (3) a “Negative” white matter group with no diffuse gliosis or focal periventricular necrosis in the cerebral white matter [23, 24]. Demographic characteristics were compared between the PVL, DWMG and Negative groups using Wilcoxon rank sum tests for continuous variables and χ2 tests for categorical variables (Table 1). The number of gray matter sites involved with an injury, i.e., acute neuronal necrosis, neuronal loss, and gliosis, was counted for each case in each group, and plotted relative to postconceptional age (i.e., gestational age plus postnatal age, PCA; in weeks) to graphically depict the total number of gray matter sites that were injured in each case. To test the hypothesis that the incidence and severity (grade 2–3/3) of neuronal necrosis, neuronal loss, and gliosis in all gray matter sites analyzed varies significantly among PVL, DWMG, and Negative cases, Fisher exact tests were used. To control for the potential impact of age on these analyses, analysis of covariance of diagnosis on lesion severity were performed, controlling for PCA. Nonsignificant interaction effects between diagnosis and age were subsequently eliminated from the models. In all analyses, P < 0.05 was considered significant.
Table 1Clinicopathologic variables of the three white matter study groups for comparison of gray matter injuriesMean ± SD; median, (range) or percentP valuePVL group n = 17DWMG group n = 17Negative group n = 7Three-wayPVL versus DWMGDemographics Gestational age (weeks)32.8 ± 3.1; 34, (26–36)31.6 ± 3.8; 33, (24–36)26.5 ± 2.3; 27, (23–30)0.0030.282 Postnatal age (weeks)3.7 ± 4.1; 2.3, (0.1–15)3.4 ± 4.0; 1.2, (0.1–12.0)0.8 ± 1.2; 0.1, (0.1–3.0)0.141 Postconceptional age (weeks)36.5 ± 5.4; 35.5, (26–52)34.8 ± 4.5; 35.0, (28.4–48.0)27.3 ± 2.8; 28.3, (23.1–30.3)0.0010.293 Length of ICU stay (days)13.8 ± 15.9; 9.5, (0–56) 23.8 ± 34.6; 5.0, (1–96) 5.0 ± 8.9; 1.5, (0.04–23.0) 0.419 Race: Caucasian8/17; 47%9/17; 53%5/7; 71%0.801 African-American2/17; 12%2/17; 12%1/7; 14% Unknown7/17; 41%6/17; 35%1/7; 14% Percent male9/17, 53%7/17, 41%4/7, 57%0.702Postmortem interval (h)16.0 ± 7.7; 18, (2–30)23.4 ± 19.6; 17, (2–72) 36.3 ± 42.4; 22, (16–132)0.364Birth weight (g)1576 ± 717; 1380, (780–2700)1832 ± 1048; 2100, (610–3800)925 ± 408; 930, (440–1400)0.232Brain weight (g)245.0 ± 81.1; 257.5, (110–340)255.0 ± 72.1; 245, (124–380) 127.6 ± 40.7; 140.0, (63.8–178.0)0.0030.905Body weight (g)2423.1 ± 1165.0; 2225, (760–4875)3276.2 ± 4706.0; 2290, (1150–21300)947.9 ± 359.2; 915, (440–1400)0.0020.815Body length (cm)42.0 ± 7.8; 41.5, (26–56)43.2 ± 4.4; 44.0, (36–51)34.7 ± 4.0; 32.5, (30–41)0.0070.589Twin or other multiple gestation3/17, 18%4/17, 24%2/7, 29%0.824Multiple congenital anomaliesa4/17, 24%2/17, 12%3/7, 43%0.242Cesarean section10/17, 59%12/17, 71%3/7, 43%0.436Cardiorespiratory factorsApgar score at 1 min4.1 ± 2.4; 4, (1–8) 4.7 ± 2.9; 5, (0–9) 3.5 ± 2.2; 3, (1–7) 0.691Apgar score at 5 min5.2 ± 2.9; 6, (1–9) 6.6 ± 2.9; 7, (0–10) 5.0 ± 2.8; 5.5, (1–8) 0.405Acute respiratory distress syndrome7/17, 41%10/17, 59%5/7, 71%0.344Mechanical ventilation13/17, 77%13/17, 77%7/7, 100%0.359Length of mechanical ventilation (days)8.4 ± 11.6; 3.0, (0–38)3.5 ± 6.3; 1.0, (1–19) 4.4 ± 8.2; 1.0, (0.04–23.0) 0.383Extracorpeal membrane oxygenation1/17%, 6%0/17%, 0%0/7, 0%0.485Continuous positive airway pressure0/17, 0%3/17, 18%1/7, 14%0.202Cardiopulmonary resuscitation any time during hospitalization5/17, 29%7/17, 41%5/7, 71%0.165Congenital heart diseasea2/17, 12%2/17, 12%1/7, 14%0.983Infectious/inflammatory factorsPneumoniaa4/17, 24%2/17, 12%0/7, 0%0.303Maternal fever at delivery0/15, 0%2/9, 22%0/4, 0%0.103Maternal history of urinary tract infection 0/15, 0%0/10, 0%1/4, 25%0.039NAChorioamnionitis1/15, 7%2/10, 20%1/5, 20%0.562Necrotizing enterocolitisa1/17, 6%4/17, 24%2/7, 29%0.415Clinical diagnosis of sepsis8/17, 47%8/17, 47%3/7, 43%0.980If a three-way P value was significant (P < 0.05), a two-way P value between the PVL and DWMG groups was performed. NA, not applicable since the incidence was 0 in the PVL and DWMG groups NS, not significanta diagnosed at autopsy
Results
Clinical and autopsy data
Seventeen cases (41%) fulfilled the criteria for PVL, while 17 cases (41%) had DWMG, and there were 7 so-called Negative cases (17%).
Pregnancy, labor and delivery
The Negative group was significantly younger in terms of gestational age (GA) than the PVL and DWMG groups, but there was no significant difference in GA between the PVL and DWMG groups (Table 1). In terms of PCA, the PVL and DWMG groups were significantly older than the Negative group (Table 1). The Negative group had significantly lower brain weight and body weight, and shorter body length, but not birth weight, than the PVL and DWMG groups. The low somatic and brain measurements in the Negative group compared to the PVL and DWMG groups reflect the early gestational age at birth and younger postnatal age at death (Table 1). The incidence of various clinical variables, e.g., chorioamnionitis, maternal fever at delivery, history of Cesarean section, and congenital anomalies were not significantly different among the three groups. The mean Apgar scores were less than seven at 1 and 5 min in all three groups (Table 1).
Syndromes
Twenty-four percent of cases had a constellation of findings classified as a genetic/developmental syndrome, e.g., Treacher-Collins syndrome, Potter’s sequence, and osteogenesis imperfecta in three PVL cases; Down’s syndrome in a DWMG case; and Fryns syndrome, Beckwith-Wiedemann syndrome, and VACTERL association in three Negative cases. Excluding these cases from the analysis had no significant effect on the results for the different acquired lesions analyzed semi-quantitatively, i.e neuronal loss and gliosis (data not shown), and thus, their data were combined with that of the non-syndromic cases in the complete analysis reported below.
Postnatal period
Although there were no statistically significant differences in postnatal age (PNA) among the three groups, the median PNA was only 0.1 week in the Negative group in contrast to 2.3 weeks in the PVL group and 1.2 weeks in the DWMG group (Table 1). The younger PNA and significantly different PCA and GA lead us to limit our gray matter comparisons to those between the PVL and DWMG groups, although the data from the Negative group is reported in all of the tables for completeness. Cardiorespiratory disorders were common in all three groups, with no significant differences (Table 1). Acute respiratory distress syndrome was noted in 41% of PVL cases, 59% of DWMG cases, and 71% of Negative cases. Seventy-seven percent of PVL and DWMG cases, and 100% of the Negative cases required ventilation (Table 1). The duration of ventilation and incidence of cardiopulmonary resuscitation was not significantly different among the three groups. Infectious and inflammatory disorders e.g., pneumonia, necrotizing enterocolitis, and sepsis, occurred in all groups, and did not differ significantly among the groups (Table 1).
White matter findings
Eighty-five percent of all cases studied had diffuse gliosis in the cerebellum, and 82% in the cerebral hemispheres. Forty-one percent of the cases had PVL, and 41% had DWMG. Macroscopically evident periventricular cysts (<5 mm in diameter) were noted in one PVL case, while chalky-white necrotic foci (2–3 mm) were visible in two cases. In all of the other PVL cases (82%), necrotic foci were only detected microscopically, and were <1 mm in diameter. Necrotic foci were typically found within a few millimeters from the ventricles. All cerebral lobes demonstrated a similar incidence of PVL (23–28%), except for the temporal lobe (12%). DWMG particularly involved the internal capsule (67%) and corpus callosum (64%) (Table 2). Necrotic foci were also identified in nonperiventricular regions, notably the internal capsule (19%) and corpus callosum (9%) (Table 2). The incidence of PVL increased with age, but only significantly so in the frontal lobe (Table 2). In contrast, the incidence of DWMG significantly increased in almost all sites with increasing gestational and postnatal ages (Table 2). By 37+ weeks, DWMG was present in 100% of the cases in the fronto-parieto-temporal lobes, 90% of the cases in the cerebellum, and 80–86% of the cases in the internal capsule and corpus callosum (Table 2). The degree of severity of DWMG significantly increased with both GA and PNA in all cases in the frontal, parietal, temporal, occipital, and cerebellar white matter (P < 0.04; data not shown). Moreover, the coefficients in the regression models for GA and PNA were similar, indicating that the degree of gliosis increased the same amount for each extra week of gestation and for each extra week of postnatal life (data not shown). In effect, the degree of gliosis increased constantly with PCA (gestational age plus postnatal age).
Table 2Distribution of white matter lesions in 41 autopsied premature infants by anatomic site and postconceptional age (weeks)White matter siteOverall incidenceIncidence by postconceptional age (weeks) P value Logistic Regression of incidence and postconceptional weeks23–2930–3637+Frontal lobePVL11/40 (28%)0/8 (0%)6/22 (27%)5/10 (50%)0.025DWMG31/40 (78%)2/8 (25%)19/22 (86%)10/10 (100%)0.004Temporal lobePVL4/33 (12%)0/6 (0%)3/22 (14%)1/5 (20%)NSDWMG22/33 (67%)1/6 (17%)16/22 (73%)5/5 (100%)0.006Parietal lobePVL7/28 (25%)0/6 (0%)6/16 (38%)1/6 (17%)NSDWMG20/28 (71%)1/6 (17%)13/16 (82%)6/6 (100%)0.010Occipital lobePVL8/35 (23%)1/5 (20%)5/23 (22%)2/7 (29%)NSDWMG28/35 (80%)3/5 (60%)19/23 (83%)6/7 (86%)NSCorpus callosumPVL2/22 (9%)0/3 (0%)1/14 (7%)1/5 (20%)NSDWMG14/22 (64%)0/3 (0%)10/14 (71%)4/5 (80%)0.049Internal capsulePVL5/27 (19%)0/2 (0%)3/18 (17%)2/7 (29%)NSDWMG18/27 (67%)0/2 (0%)12/18 (67%)6/7 (86%)NSCerebellumPVL3/40 (8%)0/8 (0%)2/22 (9%)1/10 (10%)NSDWMG34/40 (85%)4/8 (50%)21/22 (95%)9/10 (90%)0.012P values denote significant differences in the incidence of DWMG with postconceptional age.NS, not significant; PVL, periventricular leukomalacia; DWMG, diffuse white matter gliosis in the cerebral and cerebellar hemisphere
To exclude the possibility that myelination glia were scored along with reactive astrocytes in these infants immunofluoresence staining was performed to co-localize O4 a marker of the developing oligodendrocyte, and GFAP, an astrocytic marker. No co-localization of these proteins was identified in of the three groups of cases studied (Fig. 1).
Fig. 1Immunoflourescence images of parieto-occipital white matter from a PVL case at 39 postconceptional weeks. O4 labeling (a) is shown in red and GFAP labeling (b) is shown in green. Merged images (c) show no co-localization of O4 and GFAP suggesting two distinct cell populations, i.e. GFAP-positive astrocytes and O4- positive oligodendrocyte precursors, are present. The scale bar represents 50 μm
Gray matter lesions associated with PVL in the premature infant
Acute neuronal necrosis
Acute neuronal necrosis, which is considered a marker of terminal/agonal injury, was common, and occurred diffusely across gray matter regions in all three groups. Sixty-six percent of PVL, 59% of DWMG, and 43% of Negative cases had two or more gray matter sites with acute neuronal necrosis (data not shown). Significant differences in the incidence of acute neuronal necrosis between the three groups were noted only in the cerebellar cortex (53%, PVL; 13%, DWMG; 0%, Negative; P = 0.008) and frontal cortex (56%, PVL; 41%, DWMG; 0%, Negative; P = 0.039). The incidence of neuronal necrosis was not significantly different at any gray matter site when adjusted for GA and PNA (data not shown).
Neuronal loss and gliosis
Neuronal loss and gliosis, considered markers of subacute and chronic injury, were more prevalent and of greater severity in PVL cases compared to non-PVL cases (DWMG and Negative groups) (Figs. 2 and 3, Tables 3 and 4). PVL cases showed more damage to the deep nuclear structures than was encountered in non-PVL cases. In PVL cases, the thalamus and globus pallidus had significantly higher incidences of neuronal loss (38 and 33%, respectively) and more severe neuronal loss (38 and 33%, respectively) than did the DWMG and Negative groups (both, 0%) (Fig. 2, Table 3). The incidence of gliosis was also significantly higher in the thalamus (56%), caudate (60%), putamen (50%) and globus pallidus (60%) in PVL than in DWMG (12–47%) and Negative cases (0–14%). The cerebellar dentate nucleus showed a significantly higher incidence of neuronal loss in PVL (29%) compared to the DWMG (6%) and Negative (14%) groups. PVL cases (29%) had significantly more severe neuronal loss in the cerebellar dentate compared to the DWMG and Negative groups (both, 0%) (Table 3). Gliosis of the basis pontis was seen in 100% of PVL cases and only 79% of DWMG and 29% of Negative cases (P = 0.001; Table 4). The hippocampus also had substantial neuronal loss and gliosis (Tables 2 and 3). PVL cases showed relatively mild cerebral cortical neuronal loss, compared to other neuroanatomic sites, while the incidence of gliosis ranged from 20% (temporal cortex) to 31% (frontal cortex) (Tables 3 and 4). By contrast, the cerebral cortex in all lobes from DWMG and Negative cases was totally free of neuronal loss and was infrequently gliotic (all <10%) (Tables 3 and 4).
Fig. 2Photomicrographs from thalami illustrating neuronal loss scores of 0 (a), 1 (b), 2 (c) and 3 (d). The asterisk in panel b denotes a focal area of neuronal loss. The scale bar represents 20 μmFig. 3Photomicrographs from the inferior olivary nuclei depicting gliosis scores of 0 (a), 1 (b), 2 (c) and 3 (d). Arrows in panels b and c indicate some of the reactive astrocytes that are present. The scale bar represents 20 μmTable 3Incidence and severity of neuronal loss in PVL, DWMG and Negative casesOverall incidenceIncidence of severity 2–3PVLDWMGNegativeP valuePVLDWMGNegativeP valueNeuronal lossCerebral cortexFrontal cortex13% (2/16)0% (0/17)0% (0/7)0.4776% (1/16)0% (0/17)0% (0/7)0.575Temporal cortex0% (0/15)0% (0/13)0% (0/6)1.0000% (0/15)0% (0/13)0% (0/6)1.000Parietal cortex8% (1/13)0% (0/11)0% (0/6)1.0008% (1/13)0% (0/11)0% (0/6)1.000Occipital Cortex0% (0/15)0% (0/16)0% (0/4)1.0000% (0/15)0% (0/16)0% (0/4)1.000Deep gray nucleiThalamus38% (6/16)0% (0/17)0% (0/7)0.00538% (6/16)0% (0/17)0% (0/7)0.005Hypothalamus20% (2/10)0% (0/10)0% (0/2)0.56710% (1/10)0% (0/10)0% (0/2)1.000Caudate13% (2/15)0% (0/16)0% (0/7)0.32913% (2/15)0% (0/16)0% (0/7)0.329Putamen13% (2/16)0% (0/17)0% (0/7)0.47713% (2/16)0% (0/17)0% (0/7)0.477Globus pallidus33% (5/15)0% (0/15)0% (0/6)0.02833% (5/15)0% (0/15)0% (0/6)0.028Cerebellum and relay nucleiBasis pontis21% (3/14)0% (0/14)0% (0/7)0.20614% (2/14)0% (0/14)0% (0/7)0.341Inferior olive15% (2/13)8% (1/13)20% (1/5)0.8078% (1/13)8% (1/13)20% (1/5)0.549Cerebellar cortex24% (4/17)6% (1/16)14% (1/7)0.44924% (4/17)6% (1/16)14% (1/7)0.449Dentate29% (4/14)0% (0/15)0% (0/6)0.03129% (4/14)0% (0/15)0% (0/6)0.031Limbic structuresHippocampus33% (5/13)0% (0/14)14% (1/7)0.05533% (5/15)0% (0/14)14% (1/7)0.055Amygdala0% (0/6)0% (0/3)0% (0/2)1.0000% (0/6)0% (0/3)0% (0/2)1.000Substantia inominata29% (2/7)0% (0/3)0% (0/1)1.00029% (2/7)0% (0/3)0% (0/1)1.000Brainstem14% (2/14)0% (0/14)0% (0/5)0.62914% (2/14)0% (0/14)0% (0/5)0.629PVL, periventricular leukomalacia; DWMG, diffuse white matter gliosis in the cerebral and cerebellar hemisphere. P values denote differences in the incidence or severity of neuronal loss at these neuroanatomic sites between PVL and DWMG groups with postconceptional ageTable 4Incidence and severity of gliosis in PVL, DWMG and Negative casesOverall incidenceIncidence of severity 2–3PVLDWMGNegativeP valuePVLDWMGNegativeP valueGliosisCerebral cortexFrontal cortex31% (5/16)6% (1/17)0% (0/7)0.10213% (2/16)0% (0/17)0% (0/7)0.477Temporal cortex20% (3/15)8% (1/13)0% (0/6)0.4950% (0/15)0% (0/13)0% (0/6)1.000Parietal cortex23% (3/13)9% (1/11)0% (0/6)0.4998% (1/13)9% (1/11)0% (0/6)1.000Occipital cortex27% (4/15)0% (0/16)0% (0/4)0.05413% (2/15)0% (0/16)0% (0/4)0.395Deep gray nucleiThalamus56% (9/16)18% (3/17)14% (1/7)0.03119% (3/16)0% (0/17)14% (1/7)0.161Hypothalamus40% (4/10)10% (1/10)50% (1/2)0.26420% (2/10)0% (0/10)50% (1/2)0.130Caudate60% (9/15)19% (3/16)14% (1/7)0.02813% (2/15)6% (1/16)14% (1/7)0.659Putamen50% (8/16)12% (2/17)14% (1/7)0.04419% (3/16)0% (0/17)0% (0/7)0.130Globus Pallidus60% (9/15)47% (7/15)0% (0/6)0.04020% (3/15)7% (1/15)0% (0/6)0.492Cerebellum and relay nucleiBasis pontis100% (14/14)79% (11/14)29% (2/7)0.00136% (5/14)21% (3/14)14% (1/7)0.684Inferior olive92% (12/13)92% (12/13)80% (4/5)0.54962% (8/13)54% (7/13)20% (1/5)0.400Cerebellar cortex29% (5/17)6% (1/16)14% (1/7)0.25912% (2/17)6% (1/16)0% (0/7)1.000Dentate43% (6/14)13% (2/15)17% (1/6)0.17721% (3/14)0% (0/15)0% (0/6)0.125Limbic structuresHippocampus47% (7/15)7% (1/14)29% (2/7)0.05620% (3/15)0% (0/14)29% (2/7)0.143Amygdala50% (3/6)0% (0/3)0% (0/2)0.32730% (0/6)0% (0/3)0% (0/2)1.000Substantia inominata29% (2/7)0% (0/3)0% (0/1)1.0000% (0/7)0% (0/3)0% (0/1)1.000Brainstem43% (6/14)20% (3/14)20% (1/20)0.5187% (1/14)0% (0/14)0% (0/5)1.000PVL, periventricular leukomalacia; DWMG, diffuse white matter gliosis in the cerebral and cerebellar hemisphere. P values denote differences in the incidence or severity of gliosis at these neuroanatomic sites between PVL and DWMG groups with postconceptional age
Glial fibrillary acidic protein immunohistochemistry performed on sections of frontal cortex from PVL (n = 10), DWMG (n = 13) and Negative cases (n = 4) showed astrocytes of two different general morphologies (Fig. 4). Greater numbers of reactive astrocytes with abundant GFAP positive cytoplasm tended to occur in the frontal cortex of PVL cases (mean score 0.75 ± 0.22), compared to DWMG cases (0.48 ± 0.19) or (0.59 ± 0.38); however, when controlled for PCA this trend was not statistically significant. Non-reactive astrocytes were found in similar frequency among PVL, DWMG and Negative cases, and no such trend, as that noted for reactive astrocytes appeared (data not shown).
Fig. 4GFAP immunohistochemical staining of frontal cortex illustrating non-reactive astrocytes (a) with a linear GFAP-positive process that is perpendicular to glial limitans, which is at the right of this image (not depicted) and a reactive astrocyte (b), with abundant GFAP-positive cytoplasm, and an eccentrically placed, enlarged nucleus. The scale bar represents 20 μm
Discussion
This neuropathologic analysis shows clearly that gray matter abnormalities are more common in the presence of PVL than in its absence (summarized in Fig. 5). Moreover, and remarkably, neuronal loss and gliosis in the cerebral cortex and deep nuclear structures are essentially confined to those infants with PVL. Thus, not a single infant with DWMG exhibited neuronal loss in the cerebral cortex, hippocampus, and deep gray nuclei. Similarly, gliosis was very unusual in these areas in the infants with DWMG. Although the incidence and severity of lesions in this autopsy series may not be completely representative of the brain pathology in premature infants who survive beyond the perinatal period, the findings are nevertheless important to understand the neuroanatomic substrate and pathogenesis of the neurological sequelae in long-term survivors.
Fig. 5Summary diagram comparing gray matter sites with a significantly higher incidence (percentages) of neuronal loss (a) and gliosis (b) in PVL (right of panel) and DWMG (left of panel) cases. Gliosis of the cerebral and cerebellar white matter, basis pontis, brainstem tegmentum and inferior olives is depicted by small red dots, and focal, periventricular necrosis in the cerebral white matter (PVL) is denoted by a large red periventricular circle
The pathogenesis of the gray matter lesions in PVL is likely due to the same phenomena implicated in the white matter lesion. The pathogenesis of PVL likely involves cerebral ischemia-reperfusion in the respiratory compromised preterm infant, in combination with one or more infectious/inflammatory and other, yet to be defined, derangements [24]. Thus, the topographic patterns of gray and white matter damage in the premature brain likely reflect a complex interplay of the differential vulnerabilities of the regions to glutamate, free radical, and cytokine toxicity. These differential vulnerabilities appear to be based upon the maturational stage of neurons and oligodendrocytes [3, 23, 40], i.e., the targeted cell types in gray and white matter injury, respectively, and upon the developmental profiles of glutamate and cytokine receptors [9, 23] and antioxidant systems [9], as well as multiple related factors [23, 40]. The term “perinatal panencephalopathy” best describes, in our opinion, the combined gray and white matter injury delineated in this study that is typical of perinatal neuropathology of prematurity. We regard PVL as a major part of this disorder that should now be considered, we believe, in the context of total brain injury. Since the majority of patients in the PVL, DWMG, and Negative groups required mechanical ventilation of comparable durations, and showed substantial involvement by inflammatory/infectious processes, it is difficult to decipher the factors responsible for the substantial brain injury in the PVL group. It is very likely that there are specific clinical factors at work that we do not yet know, or were not analyzed in this study, e.g., lowest oxygen levels, alterations in acid-base status, and dysfunction in cerebral autoregulation, which are difficult to analyze in a meaningful way from complicated neonatal records. Thus, this study is hypothesis-generating for a prospective analysis of the key clinical factors involved in the pathogenesis of perinatal panencephalopathy.
The presence of isolated hypertrophic astrocytes in the cerebral white matter of premature infants, as reported in this series in the DWMG group, has been recognized for decades, but its significance remains unknown. Focal necrosis and diffuse hypertrophic astrocytes that are associated with “globules” and “acutely damaged glia” have been considered histological manifestations of the same disorder of immature cerebral white matter for which the term acquired perinatal telencephalopathy (PTL) has been coined [27]. Yet, hypoxic–ischemic white matter injury may follow a continuum of damage, from mild (gliosis [hypertrophic astrocytes] alone) to severe (periventricular necrosis combined with gliosis) [27]. Astrocytes, however, may also normally undergo hypertrophy in the late fetal and perinatal white matter as an obligatory developmental change, potentially due to the “physiological oxidative stress” of active myelin sheath synthesis, and thus may not be a marker of pathology at all [15]. These so-called myelination glia are immature oligodendrocytes that express markers such as O4, and are morphologically similar to GFAP positive reactive astrocytes. Coimmunofluoresence studies show no overlap in the expression of O4 and GFAP. The significant differences in age and survival encountered among the PVL, DWMG and Negative groups in this study precluded using the Negative group as a control representing “no white matter injury”. The Negative group consisted of infants who were born after significantly shorter gestational periods and who survived for significantly shorter time-periods postnatally than those in the PVL or DWMG groups. Thus, it is possible that the infants in the Negative group showed no white matter gliosis because the white matter is not vulnerable to injury at this early age, immature astrocytes are not capable of mounting a hypertrophic reaction to injury at this early time-point, and/or the patients did not survive long enough for astrocytic hypertrophy to develop. Further studies are needed to examine the significance of astrocytic hypertrophy in developmental pathology. The challenge is heightened by the unavoidable fact that live-born infants dying during the last half of gestation are not “normal”, but rather, typically die in intensive care units with multiple complications of prematurity that are known to adversely affect the brain.
This study suggests that neuronal loss and/or gliosis in the perinatal period in gray matter sites critical to cognition, memory, and learning, i.e., thalamus [7, 38], basal ganglia [33], hippocampus [7, 26], and cerebellum [21, 37], play a role in cognitive defects in long-term survivors of prematurity. The neuroanatomic structures involved with neuronal loss and/or gliosis correlates well with the neuroimaging data, which has shown volumetric deficits in the thalamus and basal ganglia [17, 18], and to a lesser degree, the hippocampus [19, 34], and cerebral cortex [35] in survivors of prematurity. This thalamic damage could be important in the pathogenesis of subsequent cognitive impairments. Of note, afferent thalamocortical axons fail to reach the cortex when the subplate neurons are ablated and abnormal cortical lamination results [10–12, 14, 22, 30]. Selective subplate neuronal loss occurs in hypoxic-ischemic injury in neonatal rats [31], underscoring the possibility of homologous injury in human premature infants [16] and the need for in depth studies of the subplate-thalamic-cortical unit in humans. Premature infants are also at high risk for cerebellar injury [6, 20, 28, 29, 32], given the mounting evidence that the cerebellum plays a role in cognition [21, 37], our finding of substantial damage in this structure and its brainstem relay nuclei suggests that it could contribute to cognitive defects in survivors. In addition, injury to the cerebellum, as well as the thalamus and basal ganglia (globus pallidus), may contribute to the motor deficits of prematurity. Traditionally, the spastic motor deficits, i.e., cerebral palsy, in preterm infants has been attributed to damage to axons in the necrotic foci in PVL that are coursing through the periventricular white matter from the motor cortex to the spinal cord [41]. Our data suggest that at least some of the common, less severe motor deficits are due to gray, as well as white, matter damage.
In conclusion, this study draws attention to the combination of white and gray matter injury in the brains of preterm infants dying in the perinatal period by the term “perinatal panencephalopathy”. Our findings suggest that future treatment strategies should target both white and gray matter damage to prevent the neurologic deficits in survivors of prematurity.

white matter gliosis
thalamus
basal ganglia
brainstem
perinatal panencephalopathy
neurodevelopmental disability
perinatal hypoxia–ischemia