Toward a better understanding of the cellular basis for cerebrospinal fluid shunt obstruction: report on the construction of a bank of explanted hydrocephalus devices

Abstract

OBJECTIVE Shunt obstruction by cells and/or tissue is the most common cause of shunt failure. Ventricular catheter obstruction alone accounts for more than 50% of shunt failures in pediatric patients. The authors sought to systematically collect explanted ventricular catheters from the Seattle Children's Hospital with a focus on elucidating the cellular mechanisms underlying obstruction. METHODS In the operating room, explanted hardware was placed in 4% paraformaldehyde. Weekly, samples were transferred to buffer solution and stored at 4°C. After consent was obtained for their use, catheters were labeled using cell-specific markers for astrocytes (glial fibrillary acidic protein), microglia (ionized calcium-binding adapter molecule 1), and choroid plexus (transthyretin) in conjunction with a nuclear stain (Hoechst). Catheters were mounted in custom polycarbonate imaging chambers. Three-dimensional, multispectral, spinning-disk confocal microscopy was used to image catheter cerebrospinal fluid-intake holes (10× objective, 499.2-μm-thick z-stack, 2.4-μm step size, Olympus IX81 inverted microscope with motorized stage and charge-coupled device camera). Values are reported as the mean ± standard error of the mean and were compared using a 2-tailed Mann-Whitney U-test. Significance was defined at p < 0.05. RESULTS Thirty-six ventricular catheters have been imaged to date, resulting in the following observations: 1) Astrocytes and microglia are the dominant cell types bound directly to catheter surfaces; 2) cellular binding to catheters is ubiquitous even if no grossly visible tissue is apparent; and 3) immunohistochemical techniques are of limited utility when a catheter has been exposed to Bugbee wire electrocautery. Statistical analysis of 24 catheters was performed, after excluding 7 catheters exposed to Bugbee wire cautery, 3 that were poorly fixed, and 2 that demonstrated pronounced autofluorescence. This analysis revealed that catheters with a microglia-dominant cellular response tended to be implanted for shorter durations (24.7 ± 6.7 days) than those with an astrocyte-dominant response (1183 ± 642 days; p = 0.027). CONCLUSIONS Ventricular catheter occlusion remains a significant source of shunt morbidity in the pediatric population, and given their ability to intimately associate with catheter surfaces, astrocytes and microglia appear to be critical to this pathophysiology. Microglia tend to be the dominant cell type on catheters implanted for less than 2 months, while astrocytes tend to be the most prevalent cell type on catheters implanted for longer time courses and are noted to serve as an interface for the secondary attachment of ependymal cells and choroid plexus.

Keywords: CSF = cerebrospinal fluid; GFAP = glial fibrillary acidic protein; HBHS = HEPES-buffered Hanks solution; HIPAA = Health Insurance Portability and Accountability Act; IHC = immunohistochemical; IRB = institutional review board; PDMS = poly(dimethylsiloxane); PFA = paraformaldehyde; RT = room temperature; SCH = Seattle Children's Hospital; TTR = transthyretin; astrocyte; hydrocephalus; microglia; shunt failure; shunt obstruction; ventriculoperitoneal shunt.

FIG. 1

Graph showing number of operative cases resulting in explanted hardware per month compared with hardware included in study. Figure is available in color online only.

FIG. 2

Gross photograph of explanted Medtronic Ares antibiotic-impregnated catheter (upper) alongside 10× images of the same CSF intake holes (lower). Note the presence of a robust astrocyte-dominant response (green) by microscopic examination as well as the relative paucity of attached cells on the surfaces between the CSF intake holes. All images obtained with 499.2-μm z-stack, 2.4-μm step size. Figure is available in color online only.

FIG. 3

Montage of images of a Bugbee wire–exposed catheter demonstrating scant or irregular IHC labeling within the lumen and CSF intake holes (arrow). Cell masses demonstrate the return of typical immunoreactivity beyond a variable distance (range 0.1–2.0 mm) from the CSF intake hole (arrowhead). All images obtained with 10× objective, 499.2-μm z-stack, 2.4-μm step size. Figure is available in color online only.

FIG. 4

Immunohistochemical images of individual CSF intake holes from 3 different explanted ventricular catheters demonstrating representative astrocyte-dominant, microglia-dominant, and mixed responses. All images obtained with 10× objective, 499.2-μm z-stack, 2.4-μm step size. Figure is available in color online only.

FIG. 5

Immunohistochemical images of a CSF intake hole that demonstrate an astrocyte-dominant response (green). Peripherally bound to the astrocyte cell mass are cells demonstrating punctate TTR (purple) labeling in the absence of GFAP or Iba-1 labeling, which are presumed to be sloughed ependymal cells (arrowheads). 10× objective, 499.2-μm z-stack, 2.4-μm step size. Figure is available in color online only.

FIG. 6

A Medtronic Ares antibiotic-impregnated catheter demonstrating extensive choroid plexus attachment via an interface with astrocytes and microglia directly bound to the catheter surface. A: In the gross photograph of the explanted catheter, choroid plexus can be seen attached to the 2 most distal CSF intake holes. The attached choroid plexus was wrapped around the catheter near the most proximal CSF intake hole when it was received from the operating room, but during catheter processing it became clear that there was no point of fixation securing it in that position. Arrows indicate which hole is being imaged in the subsequent figure panels. B: A maximum projection 10× field (408-μm z-stack, 2.4-μm step size) of the distal-most CSF intake hole. The edges of the CSF intake hole were identified by means of bright-field imaging and are indicated by a dashed white line. Note the presence of clusters of astrocytes (arrow and double arrow) as well as a mixed cluster of microglia and astrocytes (arrowhead) at the perimeter of the CSF intake hole. C: To better appreciate the nature of the cell-catheter interface, the entire maximum projection from panel B has been vertically rotated 84°, as has the dashed white line denoting the CSF intake hole edges. The low resolution of the rotated 3D projection reflects decreased resolution in the z-dimension (2.4-μm/pixel as opposed to 0.8-μm/pixel in the x-y plane). Video 1 demonstrates this 3D rotation as an 8.4-second movie. The white brackets on the right of the panel denote the selected portions of the z-stack presented as maximum projections in panels B, D, and E. The arrow, double arrow, and arrowhead denote the same astrocyte and microglia/astrocyte clusters highlighted in panel B. D: A maximum projection of 100 z-stacks (237.6-μm) above the confines of the CSF intake hole, allowing for visualization of the choroid plexus (red) morphology. Note the presence of some astrocytes (green) attached to the surface of the lobule of choroid at the 3 o’clock position. E: A maximum projection of the 51 z-stacks (120.0-μm) at the CSF intake hole interface. Note that at the interface with the catheter surface, astrocytes (green) and, to a lesser extent in this case, microglia (blue) predominate, with the 2 astrocyte clusters (arrow and double arrow) and 1 mixed microglia/astrocyte cluster (arrowhead) appearing to serve as points of choroid plexus (red) fixation to the catheter. F: A maximum projection 10× field (499.2-μm z-stack, 2.4-μm step size) of the second most distal CSF intake hole demonstrating an extensive mesh of astrocytes (green) at the CSF intake hole interface, which extends onto the large mass of choroid plexus (red) above the confines of the CSF intake hole (3–7 o’clock positions; hole edges denoted by dashed white line). A cuff of choroid is seen to extend just outside the confines of the CSF intake hole (11–3 o’clock positions). G: Although not macroscopically visible in panel A, a small piece of choroid plexus (red) was noted at the time of catheter imaging within the depths of the third most distal CSF intake hole. Given its depth within the hole, the catheter had to be sectioned to completely visualize the interface between the mass of choroid plexus and the catheter surface. The dashed white lines denote the cross-sectioned boundaries of the CSF intake hole. Note that again astrocytes predominate at the interface with the catheter surface. Maximum projection, 10× objective, 360-μm z-stack, 2.4-μm step size. Figure is available in color online only.