Malfunctioning central venous catheters in children: a diagnostic approach

Central venous access is increasingly becoming the domain of the radiologist, both in terms of the insertion of central venous catheters (CVCs) and in the subsequent management of these lines. This article seeks to provide an overview of the CVC types available for paediatric patients and a more detailed explanation of the spectrum of complications that may lead to catheter malfunction. A standard catheter contrast study or ‘linogram’ technique is described. The normal appearances of such a study and a detailed pictorial review of abnormal catheter studies are provided, together with a brief overview of how information from catheter investigations can guide the management of catheter complications.
Introduction
Central venous access forms a vital part of the management of many paediatric conditions. For many years, tunnelled central venous catheters were largely reserved for patients on chemotherapy regimens or long-term total parenteral nutrition (TPN), but more recently, reliable central venous access has proved central to the management of an extensive range of conditions such as those requiring long-term antibiotic therapy, chemotherapy or haemodialysis. The emergence of image-guided vascular access techniques has brought central venous access increasingly into the domain of interventional radiology (IR), with perceived advantages of shorter operating times, an increased likelihood of achieving access in difficult cases, fewer procedural complications, lower operating costs, and a probable improvement in long-term venous patency rates [1–10]. This shift from general surgery to radiology has, however, brought with it an increased demand on radiology departments to maintain and manage indwelling central venous catheters (CVCs). In children, meticulous attention to catheter position is vital to ensure that CVCs are kept functioning for as long as possible in order to minimize the number of catheter replacements a child may have to undergo. In some practices, malfunctioning catheters are removed and replaced without investigation; in other centres, including our institution, it is recognized that there are a number of interventions available to salvage blocked or displaced catheters, allowing alternative access sites to be preserved for future use. Plain radiographs and fluoroscopic contrast examinations are central to the radiological investigation of malfunctioning catheters [11, 12]. Many radiologists, however, may be perplexed by the increasingly wide variety of venous access devices available and unaware of some of the potential CVC-related complications that develop in children or the catheter-salvage techniques available.
This article gives a brief overview of the central venous access devices available and the potential causes of catheter malfunction. A standard catheter contrast study or ‘linogram’ technique is described. The normal appearances of such a study and a detailed pictorial review of abnormal catheter studies are provided, together with a brief overview of how information from catheter investigations can guide the management of catheter complications.
Background
Central venous access is required for the safe delivery of a number of drugs and other intravenous therapies into large-calibre vessels, to allow adequate dilution of the drug and avoid vessel irritation. Here the term ‘central’ is taken to encompass the brachiocephalic veins, the superior vena cava (SVC), the suprahepatic inferior vena cava (IVC) and the right atrium (RA). A centrally placed catheter with a reasonable-size lumen also allows regular noninvasive blood sampling, which has significant benefits for both the clinical team and the patient.
There is much debate surrounding the issue of the ideal CVC tip position and a detailed presentation of this argument is beyond the remit of this article. Suffice to say that the argument that a catheter tip position at the level of the RA is unsafe due to the risk of myocardial perforation and subsequent tamponade is increasingly unsupported in modern practice with the advent of softer catheter tips, with the probable exception of 2F catheters, traditionally inserted by the cot-side as neonatal long lines, which have a slightly stiffer tip. There is much in the literature regarding cardiac perforation by 2F neonatal long lines in low-birth-weight infants and it seems generally accepted that these catheters are safer when positioned outside the pericardial reflections [13–20]. This paper, however, deals specifically with radiologically or surgically inserted Silastic (silicone elastomer) catheters of 4F calibre and above. It is now recognized that these larger-calibre catheters are significantly less likely to develop tip thrombus or a fibrin sheath when left to move freely within fast-flowing blood at the level of the right atrium and are likely to function for a significantly longer period of time in this position [8, 21–25]. The position of the RA is best estimated at a level 1.5 vertebral bodies below the carina on a chest radiograph [26, 27]. It is worth remembering, however, that the position of a catheter tip will vary widely with patient positioning, particularly in children, and should be evaluated with caution on any image [8, 22, 28–30].
Catheter types
There is a vast array of central venous access devices available for paediatric use; particular devices may be preferred in different centres. In general, however, catheter subtypes can be defined according to their site of insertion, whether they are placed via a subcutaneous tunnel or, with particular reference to haemodialysis catheters, according to function. To achieve a central tip position, a catheter can be inserted using a tunnelled or a nontunnelled technique. Tunnelled devices are usually inserted via access to a neck vein and are tunnelled to that site through the adjacent subcutaneous tissues, exiting the skin at an appropriate position on the chest wall. The tunnelled, extravascular portion of the catheter is considered to provide a barrier to ascending infection and with time will adhere to the subcutaneous tissues, decreasing the likelihood of catheter displacement. This adherence is encouraged by the presence of a short Dacron cuff attached to the catheter, which causes a focal region of fibrosis or ingrowth in the track and thus provides further anchorage for the catheter (Fig. 1). Insertion of such catheters almost always requires general anaesthesia in children to allow safe venous puncture. The vein puncture itself can be performed using a percutaneous, US-guided technique or a surgical open venotomy. Removal of a cuffed catheter that has been in situ for longer than a week or so generally also requires a general anaesthetic in children, as removal or dissection of the cuff can be traumatic. Hence, tunnelled CVCs are best suited for medium- to long-term venous access requirements such as chemotherapy and TPN regimens. Tunnelled catheters range in calibre from 2.7F to 12F and have a single or double lumen. Haemodialysis catheters are very similar to other double-lumen catheter devices. The lumens are generally of a larger calibre, however, to allow high flow rates for efficient dialysis and the end holes or tips may be staggered (Fig. 2).
Fig. 1A 4F single-lumen Hickman (Bard Access Systems, Salt Lake City, Utah) catheter for tunnelled central venous access. Inset illustrates the Dacron ingrowth cuffFig. 2A permanent (tunnelled) haemodialysis catheter with staggered tip for optimal flow rates (12.5F Vas-Cath Soft Cell; Bard Access Systems, Salt Lake City, Utah)
An alternative to a traditional tunnelled CVC is a totally implanted CVC or implanted venous access device (iVAD), commonly known as a portacath. This comprises a reservoir attached to a standard Silastic catheter (Fig. 3). The catheter is inserted using a standard tunnelled catheter technique. The reservoir is buried within the subcutaneous tissues of the chest wall, so that the device is completely implanted. When required, the reservoir is accessed percutaneously via a specialised noncoring needle. The reservoir can be accessed up to 1,000 times before replacement is needed. Such buried devices have a lower infection rate than externalized catheters and allow greater freedom for social activities such as swimming, but require patients to undergo repeated needle access procedures. They are best suited for intermittent therapies such as enzyme infusions in metabolic disorders, factor infusions in haemophilia or recurrent antibiotic requirements for conditions such as cystic fibrosis.
Fig. 3A totally implantable port (Bard Access Systems, Salt Lake City, Utah) with reservoir and 6.6F catheter. Inset illustrates the specialized noncoring port access needle
Nontunnelled CVCs are placed directly into a vein via a skin incision overlying the vein and are simply sutured to the adjacent skin (Fig. 4). Access sites include the neck and the extremities. Nontunnelled neck lines are commonly placed for complex anaesthesia cases or in the intensive care setting. The neck veins can also be used for short-term dialysis catheters in patients who do not require a longer-term tunnelled device. For longer-term indications for a nontunnelled line, operators usually prefer to access venous sites in the extremities. Small-calibre (4–5F) peripherally inserted central catheters (PICCs) are usually placed via the veins of the upper arm (Fig. 5). In a cooperative child they can often be placed via a peripheral venous cannula, without sedation or general anaesthesia, and are most useful for providing reliable access for medium-term therapies in children. The catheter is advanced to the level of the central vessels using fluoroscopic guidance in the radiology department or a non-image-guided ‘best-estimate’ technique at the bedside. These catheters provide reliable venous access for indications such as extended antibiotic treatment in patients with osteomyelitis or infected neurosurgical shunts and for repeated blood-sampling in needle-phobic patients, or children in whom peripheral access is becoming technically difficult. PICCs are also increasingly used for longer-term therapies such as chemotherapy, pulmonary hypertension and TPN, and can function for a year or more. Single-lumen peripherally inserted CVCs or PICCs are commonly 4F in calibre; this allows more reliable function than the 2F neonatal long lines, particularly for blood sampling (aspiration via a catheter requires a faster flow rate in the vessel being sampled, and is therefore more sensitive to lumen calibre when compared with infusion through the same catheter). Double-lumen 5F catheters are also widely used, although each lumen is small and hence these catheters, although useful for multidrug therapies, are less reliable for blood sampling. Some types of PICC are now approved for CT contrast medium infusions via a pump injector.
Fig. 4A triple-lumen 7F catheter (Arrow-Howes; Arrow International, Reading, Pa.) for short-term nontunnelled central venous accessFig. 5A double-lumen 5F PICC (Bard Access Systems, Salt Lake City, Utah)
Strategies in managing catheter malfunction
CVC malfunction can be approached by the clinical team and the radiologist in a number of ways. In some centres a policy of removing all malfunctioning catheters without investigating the cause of the malfunction is maintained. In most institutions, however, protocols are available for attempted thrombolysis of blocked catheters and in some units revision of displaced or incorrectly positioned lines is attempted. In our opinion, an attempt should always be made to diagnose the cause of a malfunctioning catheter, with the aim of possible catheter salvage and, therefore, a potential reduction in the number of both repeated anaesthesia and venous access attempts that a patient may have to undergo. An institutional policy regarding the management of malfunctioning CVCs is helpful and should be made with the facilities of that institution in mind. In particular, contrast studies involving radiation exposure should only be undertaken if the results of the study would alter the subsequent management of the line. In general, an IR department is more likely to be able to offer salvage procedures than a surgically managed service, due to differences in both the imaging and interventional techniques available within each speciality [3, 10, 31].
Initial examination of the catheter
It is important to examine both the catheter and its skin exit site prior to performing any radiographic investigation, so that a simple cause of catheter malfunction, such as a break in the catheter itself or wear at the clamp site, can be established. It is not uncommon to find that the skin suture of a newly placed catheter is too tight and is restricting flow.
Fluoroscopic or plain radiographic views
Plain radiographic views of the catheter should be acquired prior to any contrast-enhanced study. These can be acquired as a chest radiograph or by using low-dose fluoroscopy at the time of a planned contrast investigation. This allows misplaced catheters (Fig. 6) and catheters of an incorrect length to be identified (Fig. 7). The entire catheter must be imaged. Studies limited to the intravascular portion of the catheter will frequently miss abnormalities such as kinking of the catheter within the skin, a finding that is particularly common with portacaths (Fig. 8), and looping or twisting of a catheter in the neck (Fig. 9). More than one view may be required to confirm a kink in the catheter of a portacath; angulation of the catheter most commonly occurs at the origin of the subcutaneous tunnel, adjacent to the reservoir (Fig. 10). Occasionally, a portacath reservoir can become detached from its catheter, and children who are compulsive ‘port fiddlers’ can encourage a catheter to loop around the port chamber so that over time the intravascular portion is shortened. A catheter may be accidentally pulled back or may migrate backwards into the soft tissues of the neck, so that the tip lies outside the vein entirely. This second complication seems to occur most commonly in chubby infants with redundant tissue in the neck (Fig. 11). Despite accurate initial placement, catheter tips can flick into an incorrect position with changes in posture or intrathoracic pressure; this occurs more commonly with catheter tips placed at the level of the SVC rather than the RA. The commonest site for a catheter tip to migrate to is the ipsilateral internal jugular vein or the contralateral brachiocephalic vein (Fig. 12). Observing the catheter tip during fluoroscopic screening, even before injecting contrast medium, allows the operator to determine whether the tip is moving freely or is wedged against a vessel or chamber wall. Exaggerated movement of the catheter when in an intracardiac position is strongly suggestive of malposition of the catheter tip within the right ventricle; most operators would agree that such a line should be shortened or replaced in such a situation, even if the child is asymptomatic, due to the risk of catheter-induced arrhythmias. Fluoroscopic screening should be performed using a low pulse rate (three pulses per second) and where available, the image-grab facility should be used to minimize radiation exposure.
Fig. 6Misplaced catheter. a Fluoroscopic image of a 10-month-old male with a portacath in situ, the tip of which has been placed into the coronary sinus. b The position of the tip is confirmed by a contrast medium studyFig. 7A chest radiograph in a 13-month-old male with a permanent haemodialysis catheter, the tip of which lies at or beyond the level of the tricuspid valve causing intermittently poor flow ratesFig. 8Kinking of the catheter. a Chest radiograph of a 6-year-old male with a portacath in situ. The catheter is kinked just beyond the reservoir (arrow). b Fluoroscopic image of a femoral temporary dialysis catheter in a 15-year-old male. The catheter is kinked at the vessel entry site, causing poor flow ratesFig. 9Fluoroscopic image of a tunnelled catheter in a 2-year-old child. The catheter has twisted in the soft tissues of the neck, causing the catheter tip to be partially withdrawn (arrow)Fig. 10Kinking of the catheter. a, b AP (a) and lateral (b) views of a portacath in a 2-year-old female demonstrating sharp angulation of the proximal catheter, limiting infusion flow rates. The kink is not clearly seen on the AP viewFig. 11Chest radiograph of a 9-month-old infant with a tunnelled CVC in situ. The catheter has migrated back into the redundant soft tissues of the neck and chest wallFig. 12Migration of the catheter tip. a Chest radiograph of a 4-year-old male with a portacath in situ. The catheter tip lies in the right internal jugular vein (short arrow). Note that the proximal catheter is also kinked (long arrow). b Chest radiograph of a 2-year-old female with a tunnelled CVC in situ. The catheter tip lies in the left brachiocephalic vein
Performing a contrast study
If the initial examination of the line is unremarkable, a contrast-enhanced study (‘linogram’) should be performed. The catheter should be accessed using a sterile technique and, where possible, any heparin solution dwelling in the line should be aspirated and discarded. Inability to aspirate from the catheter should be documented, as this narrows the list of potential causes of catheter malfunction, as discussed below. If only one lumen of a multilumen catheter is malfunctioning, imaging should first be performed through the functioning lumen as this may demonstrate the underlying problem and obviate the need for access of the second lumen. Water-soluble nonionic contrast medium should be used and the operator should aim to minimize the dose of contrast medium that the child receives.
The initial contrast medium injection should be performed slowly while the entire catheter is screened so that complications anywhere along the length of the line are readily identified (Fig. 13). The operator should look for signs of pooling of contrast medium in the soft tissues surrounding the catheter. Extravasation and pooling of contrast medium in the neck are usually painful; a slow gentle injection will minimize both the patient’s discomfort and the risk of loss of the child’s confidence in the operator. When examining a malfunctioning portacath device, tangential and en face views of the port reservoir should be obtained, to ensure that it fills uniformly and does not contain thrombus (Fig. 14). If the initial slow injection of contrast medium appears normal, a controlled but forceful hand injection should then be performed to study in more detail the flow of contrast medium away from the catheter tip. Digital subtraction angiography (DSA) should be used during image acquisition where possible, to achieve a diagnostic study with one injection, minimizing both contrast agent and radiation dose. Cooperative patients should be encouraged to breath-hold. If the study is performed in an imaging suite without DSA capability, screening should be focused on the catheter tip and the fluoroscopic pulse rate may have to be increased to either 15 pulses per second or ‘continuous’ screening to obtain adequate images. Diagnostic imaging of the contrast medium jet is hardest in small patients with rapid breathing and significant cardiac motion.
Fig. 13Fluoroscopic imaging of a tunnelled catheter in a 4-month-old female. The entire catheter is imaged during slow instillation of contrast mediumFig. 14Normal portacath. a, b Tangential (a) and en face (b) views of a portacath reservoir in a 7-year-old male. Note that in both views there is normal opacification of the reservoir during contrast medium instillation
The contrast medium jet should be of the same diameter as the lumen of the catheter and should exit the catheter in a linear forward direction, rapidly moving away from the catheter tip rather than pooling at the tip. The jet should fan out appropriately to fill the lumen of the vessel or chamber (Fig. 15). The operator should be aware of the configuration of the catheter, so that a catheter with a side hole, such as the Groshong PICC (Bard Access Systems, Salt Lake City, Utah) is not misinterpreted as a fractured or partially occluded line (Fig. 16).
Fig. 15Fluoroscopic image of contrast medium flowing freely from the catheter tip in an 8-year-old male. The contrast medium immediately fans out to fill the right atriumFig. 16Normal appearance of the contrast medium jet exiting the side hole of a PICC
It is important to remember to study both lumens of a double-lumen catheter if injection of the first lumen is normal. In patients with intermittent catheter malfunction, attempts should be made to recreate the situation in which problems occur; often, catheter function is position-dependent and placing the child in the troublesome position may demonstrate the cause of catheter malfunction. This may include a significant change in tip position following a change in posture or intermittent occlusion of the catheter lumen at the level of the clavicle due to catheter compression between the clavicle and the first rib (‘pinch-off’ syndrome).
If the child presents with symptoms of venous occlusion, such as localized pain and swelling over the venous access site or sudden swelling of the face or extremity, a formal venogram via a peripheral cannula can be performed to determine the presence of catheter-related venous thrombosis (see below).
Once the study is completed, the catheter should always be flushed and, where required, ‘locked’ with heparin solution of an appropriate concentration. This should be documented in the clinical notes as part of the examination. If the catheter is deemed unsafe for use, this too should be clearly documented.
Interpreting an abnormal contrast study
Once contrast medium has been instilled, a careful examination of the entire length of the catheter should be performed. Abnormal findings may be subtle, such as narrowing of the lumen at the site of the skin suture or clamp (Fig. 17). Contrast medium will leak into the soft tissues at the site of any break in the catheter wall (Fig. 18). In such cases the point of extravasation can often be clinically predicted by the patient’s clinical symptoms (focal swelling within the subcutaneous tunnel or leak of infusate from the catheter exit site) and the contrast agent dose therefore minimized.
Fig. 17Catheter narrowing. a Image acquired during contrast injection through a double-lumen catheter in a 7-year-old male. The catheter is damaged at the site of previous kinks or catheter clamps (arrows). b Focal narrowing of the opacified lumen of a PICC (arrow) in a 3-year-old male where the skin suture device has been attached to the catheter too tightlyFig. 18Extravasation. a Extravasation of contrast medium into the soft tissues at the site of a catheter break. b Contrast medium is seen leaking from a break in the connection between the portacath reservoir and the catheter
Inability to aspirate from a catheter that otherwise flushes well indicates one of three possible causes of catheter malfunction: the presence of a fibrin sheath, catheter tip thrombus, or position of the catheter tip against a vessel or chamber wall. All three causes create a valve effect at the catheter tip, so that the obstruction is temporarily alleviated by forward pressure during infusion but recurs with negative pressure on attempted aspiration (Fig. 19).
Fig. 19Line drawing demonstrating the effect of a fibrin sheath. a Flushing the catheter displaces the sheath or thrombus from the catheter tip and allows saline to exit the lumen. b Attempted aspiration sucks the sheath back onto the catheter tip, causing withdrawal occlusion
Gentle contrast medium injection through a catheter that is resting against a vessel or chamber wall will demonstrate rapid but asymmetric or oblique flow of contrast agent away from the catheter tip (Fig. 20). This appearance should be distinguished from an irregular jet due to tip thrombus (see below). It is vital to identify this problem during an initial slow instillation of contrast medium as a forceful injection may damage the vessel wall.
Fig. 20Fluoroscopic contrast study of a haemodialysis catheter in a 9-year-old female. The lumen with a more distal tip has been opacified. The catheter is too long and the distal tip lies up against the inferior wall of the right atrium, causing the contrast jet to be deflected
A fibrin sheath or sleeve thrombus is caused by build-up of fibrin material around the intravascular portion of the catheter. This phenomenon may occur in the majority of catheters, as early as 24 h after catheter insertion [8, 32, 33]. Fibrin sheaths cause malfunction only when the sheath extends around or over the tip of the catheter. A complete sheath prevents any contrast medium from flowing away from the catheter tip; instead it tracks retrogradely along the intravascular portion of the catheter, between the catheter and the fibrin sheath, and then spills into the soft tissues at the point of venous access (Fig. 21). In such cases, the patient will present with symptomatic swelling at the venous access site, usually the neck, during attempted catheter flushes. It is essential to identify initial contrast medium pooling at the catheter tip in the early phase of the injection followed by tracking of contrast medium back along the length of the catheter on later images, so that this is not misinterpreted as extravasation due to catheter fracture. To ensure a correct diagnosis in such cases, sequential images should be acquired or ‘grabbed’ during a very slow injection. In some cases, the fibrin sheath may be very thin and it is broken or fenestrated during flushing of the catheter. A circumferential but incomplete fibrin sheath will cause narrowing of the contrast jet as it exits the catheter, with delayed fanning out of contrast medium. This is a subtle, but classic, sign of a fibrin sheath (Fig. 22). The operator must ensure that the contrast agent jet is of a similar diameter to that of the catheter lumen, so that a circumferential thrombus or fibrin sheath causing partial occlusion of the end hole is not missed.
Fig. 21Fibrin sheaths. a, b Contrast medium is instilled into a tunnelled catheter in a 4-month-old child. The catheter has been partially pulled back and the tip lies within the left brachiocephalic vein. A fibrin sheath occludes the catheter. Instead of flowing away from the catheter tip, contrast medium tracks back along the side of the catheter (aarrow) and extravasates into the tissues of the neck (b). c Contrast study performed in a 2-year-old child with a catheter occluded by a fibrin sheath. Contrast medium tracks back along the catheter and into the subcutaneous tunnel, outlining the cuff, as shown diagrammatically in the line drawingFig. 22Fibrin sheaths. a Catheter contrast study in a 5-year-old male. Contrast medium exits the catheter tip in a linear manner, but the contrast jet is significantly narrower than the lumen of the catheter (arrow), implying the presence of a circumferential, nonocclusive fibrin sheath. The effect of the fibrin sheath is shown diagrammatically. b Contrast medium exits the tip of a portacath in an 11-year-old male. The jet fails to fan away from the tip (arrow) of the catheter, instead simply filling the lumen of the sheath. c A similar appearance is seen during a contrast study of a PICC in another patient. An arrow marks the catheter tip. Note the apparent irregular calibre of the catheter itself, a sign that contrast medium is tracking back along the catheter as shown in Fig. 21c
Catheter thrombus is most likely to occur in catheters that are inadequately or infrequently flushed, or that are sited in small vessels with a low volume and rate of blood flow around the tip, as has occurred in the case shown in Fig. 21. Thrombus within the catheter lumen itself is seen as a filling defect in the catheter during injection (Fig. 23). Tip thrombus causes irregularity of the jet of contrast medium as it exits the catheter and may deflect the jet away from the long axis of the catheter (Fig. 24). Thrombus may extend into the vessel itself, which will cause irregular filling of the vessel beyond the catheter tip (Fig. 25).
Fig. 23Image acquired during contrast medium injection through one lumen of a double-lumen catheter in a 2-year-old female. There is a filling defect within the catheter (arrow) at the site of a focal intraluminal thrombusFig. 24Thrombus. a Fluoroscopic image acquired during a catheter contrast study in a 3-year-old female. The contrast medium jet is deflected away from the long axis of the catheter, implying the presence of partially occlusive thrombus at the catheter tip. The effect of the tip thrombus is shown diagrammatically. b Thrombus is adherent to the tip of the distal lumen of a haemodialysis catheter in a 7-year-old male. Contrast medium exits the lumen eccentrically and outlines the thrombusFig. 25Contrast study of a left femoral catheter in a 7-month-old child. The short white arrow shows the tip of the catheter. Contrast medium outlines the common iliac veins and IVC. There are several filling defects in the IVC (long white arrows) consistent with venous thrombus beyond the catheter tip. Note that contrast medium also refluxes into ascending lumbar veins on the left (black arrows)
Catheter-induced venous thrombosis is influenced by catheter dwell time, the size of the catheter relative to the vessel, the access vein used, infusate type, and the presence of any prior venous interventions or systemic comorbidities in the patient [34–38]. It is likely to be a relatively common occurrence [39], although evidence for this in the literature is skewed by variations in the diagnostic techniques and pathological definitions used in various studies. Many cases of pericatheter venous thrombosis are asymptomatic and the catheter continues to function, so this complication is likely to be under-reported. Where they occur, symptoms include localized pain and swelling overlying the accessed vein and sudden facial or extremity swelling. It is important that the clinical team rule out septic thrombophlebitis in symptomatic patients. Isolated facial or extremity swelling is, however, strong evidence for acute venous thrombosis [34]. Contrast-enhanced catheter studies will demonstrate irregular occlusion of the involved vein (see Fig. 25). In well-established cases, drainage may occur only via tortuous collateral vessels. It may be appropriate to perform a venogram of the affected vessel via a peripheral cannula rather than the catheter at this stage, to assess drainage of the extremity (Fig. 26). Some operators may prefer to perform a venogram immediately prior to placement of a new catheter, to allow time for possible recanalization of the vessel or formation of substantial collaterals in the interim period (Fig. 27).
Fig. 26Left arm venography in a 1-year-old female with a malfunctioning tunnelled catheter in situ. Contrast medium fails to fill the occluded SVC and there are irregular filling defects within the left brachiocephalic vein, consistent with mural thrombi. Small collaterals are forming in the left supraclavicular regionFig. 27Right arm venogram in an 8-year-old boy performed prior to placement of a new catheter. The study demonstrates multiple irregular venous collaterals draining the arm following occlusion of the subclavian and brachiocephalic veins
Implications for catheter salvage techniques
Diagnostic catheter studies are central to the management of malfunctioning catheters. A detailed discussion of catheter salvage techniques is beyond the remit of this review; a number of the techniques outlined below are dependent on the services of a proactive IR department. Most institutions at least have a ward-based thrombolysis protocol for occluded CVCs and these techniques play a significant role in extending the life of a malfunctioning catheter [10, 40–45].
Catheters that are found to be too long can be shortened by a variety of techniques. The simplest involves withdrawal of the intravascular portion of the catheter at the venous access site while the tunnelled portion is left in situ. The catheter is then re-measured and shortened, prior to re-puncture of the venous access site and replacement of the intravascular portion of the catheter [46, 47]. If there is sufficient excess length, the catheter can be cut when it is partially withdrawn, and the tip wired to maintain access, avoiding the need for re-puncture.
Kinks in the subcutaneous tunnel of a CVC can often be straightened by accessing either the venous access site or the skin exit site of the catheter. Intervention at the venous access site may result in an increase in the intravascular length of the catheter, a result that is beneficial in catheters that have migrated back into the tunnel or the neck.
Catheter tips that have flicked out of position can occasionally be repositioned using one of several techniques. Often a forceful injection of saline through the displaced catheter will encourage the tip to return to its original position, in the direction of flowing blood [10]. Obstinate catheters may respond to placement of a guidewire through the catheter lumen, which stiffens the catheter sufficiently to encourage repositioning of the catheter [10, 48]. An alternative technique involves venous access via a femoral approach, allowing the displaced catheter to be snared and pulled back into the appropriate vessel [10, 49–51].
Catheter tip thrombus can be dislodged by inserting a tip deflecting guidewire through the catheter and rotating the wire once it is just beyond the tip of the catheter [9, 10]. Such techniques, although effective, are rarely long lasting.
Fibrin sheath stripping techniques can be attempted in patients in whom replacement of a malfunctioning catheter is undesirable, such as those with a high risk for general anaesthesia or limited alternative access sites. Again, femoral venous access is obtained and the catheter is ensnared using a standard vascular snare device (Fig. 28). Gentle manipulation of the snare along the length of the catheter is performed in an attempt to dislodge and retrieve the fibrin sheath [9, 10, 51–55]. Symptomatic pulmonary embolism is unlikely, although the procedure is contraindicated in subjects with a right to left shunt or severe cardiopulmonary disease that will not tolerate a pulmonary embolus [32, 55].
Fig. 28The distal portion of a malfunctioning haemodialysis catheter has been snared from a femoral approach in an attempt to strip the fibrin sheath from the catheter
Implications for catheter replacement techniques
The results of catheter contrast medium studies are often valuable in planning catheter replacement. Operators are likely to alter their approach to venous access in the presence of a documented fibrin sheath or venous occlusion. Resiting a catheter into the lumen of a fibrin sheath is highly likely to result in a rapid reestablishment of the sheath and subsequent catheter occlusion. In cases where reaccess of a vessel containing a fibrin sheath is necessary, it is sensible to attempt an US-guided puncture parallel to the fibrin sheath, rather than into it (Fig. 29). Alternatively, the sheath can be deliberately cannulated so that a guidewire is placed through the sheath followed by a standard angioplasty balloon that, when inflated, encourages break-up of the sheath or incorporation of the sheath into the vessel wall [9, 53].
Fig. 29US image of the right internal jugular vein in a 12-year-old female. A linear filling defect is attached to the vessel wall (arrow), likely to represent a previous fibrin sheath in this patient with a history of multiple previous access catheters
Documentation of complete venous occlusion is vital prior to replacement of a CVC. This allows a more considered approach to catheter placement and appropriate discussion with the family prior to a difficult procedure. Recanalization and venous stenting techniques or use of unusual access sites should be considered in children who would otherwise require open surgical procedures to maintain reliable venous access.
Conclusion
Reliable central venous access is key to the management of many paediatric conditions. The insertion of central venous access devices is increasingly becoming the remit of the interventional radiologist, a paradigm shift that will require general radiologists to play some role in the subsequent management of these catheters. In institutions where the specific cause of catheter malfunction may alter subsequent management, radiologists need to be familiar with the catheter types used and their imaging appearances, both on plain radiography and on contrast investigations. Accurate diagnosis of CVC malfunction can significantly alter outcome for patients in whom repeated venous access procedures can often become a source of greater morbidity than their primary condition.

central venous catheter
children
complications


Judah Folkman, a pioneer in the study of angiogenesis

More than 30 years ago, Judah Folkman found a revolutionary new way to think about cancer. He postulated that in order to survive and grow, tumors require blood vessels, and that by cutting off that blood supply, a cancer could be starved into remission. What began as a revolutionary approach to cancer has evolved into one of the most exciting areas of scientific inquiry today. Over the years, Folkman and a growing team of researchers have isolated the proteins and unraveled the processes that regulate angiogenesis. Meanwhile, a new generation of angiogenesis research has emerged as well, widening the field into new areas of human disease and deepening it to examine the underlying biological processes responsible for those diseases.
Early evidence of tumor cells releasing specific growth factor for blood vessels
In 1939, Ide et al. [1] were the first to suggest that tumors release specific factors capable of stimulating the growth of blood vessels. In 1945, Algire and Chalkley [2] were the first to appreciate that growing malignancies could continuously elicit new capillary growth from the host. They used a transparent chamber implanted in a cat’s skin to study the vasoproliferative reaction secondary to a wound or implantation of normal or neoplastic tissues. They showed that the vasoproliferative response induced by tumor tissues was more substantial and earlier than that induced by normal tissues or following a wound. They concluded that the growth of a tumor is closely connected to the development of an intrinsic vascular network.
In 1956, Melwin and Algire [3] found that the vasoproliferative response of normal or neoplastic tissues transplanted into muscle was not significantly different with respect to the time of onset of new blood vessels, though it was stronger when the implantation was performed in a resection area. In addition, while normal tissues induced a vasoproliferative response confined to the host, tumor tissues induced the formation of neovessels that pierced the implant. Lastly, the intensity of the response seemed to be influenced by the distance between the implant and the host’s vessels: normal tissue was unable to induce a response if placed more than 50 μm away, whereas tumor tissue had a longer activity range.
In 1968, Greenblatt and Shubik [4] implanted Millipore chambers (pore size 0.45 μm) into a hamster’s cheek pouch and placed some tumor fragments around them. In a few days, the growing tumor mass engulfed the whole chamber, whose pores were permeable to the tumor interstitial fluid, but not to the tumor cells. New blood vessels, however, were formed in any case very likely through the release of a diffusible factor that could pass through the pores. Ehrman and Knoth [5] confirmed these data with tumor fragments laid on Millipore filters planted on the chick embryo chorioallantoic membrane (CAM).
Tumors in isolated perfused organs: absence of angiogenesis
In 1963, Folkman and Becker studied hemoglobin solutions as potential substitutes for blood transfusion. To test which solution was optimal for tissue survival, they perfused these solutions through the vasculature of canine thyroid glands, by using an apparatus with a silicone rubber oxygenator. The glands survived for about 2 weeks. They could distinguish different hemoglobin preparations by histologic analysis of the thyroid glands after a week or more of continuous arterial perfusion. To determine if these isolated organs could support growth, they injected them with adult mouse melanoma cells. Tiny tumors developed but stopped growing at 1- to 2-mm diameter and never became vascularized [6]. Endothelial cells swelled and could not proliferate in the presence of free hemoglobin solutions lacking platelets [7]. However, the tumors were not dead. When they were transplanted to their host mice, they rapidly vascularized and grew to more than 1 cm3.
Folkman et al. observed that when tumor cells were inoculated into isolated perfused organs, tumors were limited in size to 1–2 mm3 [6]. Subsequently, they found that neovascularization does not occur in isolated perfused organs and that tumors transplanted from these organs to syngeneic mice became vascularized and grew rapidly to 1–2 mm3. This was the first evidence that the absence of neovascularization correlated with severe restriction of tumor growth.
The data were consistent with work from Harry Green, who had shown long before that growth of rabbit tumors transplanted into the anterior chamber of the guinea pig coincided with the growth of new blood vessels. Tumors that remained viable, but did not grow, had no visible blood vessels [8].
Hypothesis: tumor growth is angiogenesis dependent
In 1971, Folkman published in the “New England Journal of Medicine” a hypothesis that tumor growth is angiogenesis dependent and that inhibition of angiogenesis could be therapeutic [9]. This article also introduced the term anti-angiogenesis to mean the prevention of new vessel sprout from being recruited by a tumor. The hypothesis predicted that tumors would be enable to grow beyond a microscopic size of 1–2 mm3 without continuous recruitment of new capillary blood vessels.
This concept is now widely accepted because of supporting data from experimental studies and clinical observations carried out over the intervening years.
Evidence that tumors are angiogenesis dependent
Folkman and collaborators provide evidence for the dependence of tumor growth on neovascularization: Tumor growth in the avascular cornea proceeds slowly at a linear rate, but after vascularization, tumor growth is exponential [10].Tumors suspended in the aqueous fluid of the anterior chamber of the rabbit eye and observed for a period up to 6 weeks remain viable, avascular, and of limited size (less than 1 mm3) and contain a population of viable and mitotically active tumor cells. These tumors induce neovascularization of the iris vessels but are too remote from these vessels to be invaded by them. After implantation contiguous to the iris, which had abundant blood vessels, the tumors induced neovascularization and grow rapidly, reaching 16,000 times the original size within 2 weeks [11]. This experiment introduced the concept of tumor dormancy brought about by prevention of neovascularization. In a parallel study, tumors were suspended in the aqueous humor of the anterior chamber, placed at various distances from the iris vessels, and compared with tumors implanted directly on the iris and with those implanted in the cornea [12]. Moving the distant, dormant tumors closer to the iris jump started their growth. This suggested that this type of tumor dormancy was caused not by cell cycle arrest or immune control, but by a lack of blood supply.B-16 mouse melanoma, V-79 Chinese hamster lung cells, and L-5178 Y murine leukemia cells were plated in soft agar [13]. After 6–7 days of incubation, spheroid colonies of 0.1 mm were visible. All spheroids first enlarged exponentially for a few days and then continued on a linear growth curve for 5–23 weeks before reaching a diameter beyond which there was no further expansion. This was termed the dormant phase. After the dormant diameter was reached, these spheroids remained viable for 3–5 months, or as long as they were frequently transferred to new medium. Cells in the periphery of the spheroid incorporated 3H-labeled thymidine while cells in the center died. This is a form of population dormancy in which the proliferating cells near the surface of the spheroid just balance those dying cells deep in the center of the spheroid.Tumors implanted on the CAM of the chick embryo do not exceed a mean diameter of 0.93 ± 0.29 mm during the prevascular phase (approximately 72 h). Rapid growth begins, however, within 24 h after vascularization, and tumors reach a mean diameter of 8.0 ± 2.5 mm by 7 days [14].Tumors grown in the vitreous of the rabbit eye remain viable but attain diameters of less than 0.50 mm for as long as 100 days. Once such a tumor reaches the retinal surface, it becomes neovascularized and within 2 weeks can undergo a 19,000-fold increase in volume over the avascular tumor [15].The CAM appears at day 5 during development of the chick embryo. The 3H-thymidine labeling index of its vascular endothelium decreases with age, with an abrupt reduction at day 11 [16]. Prior to day 11, labeling index is approximately 23%; during 11 days, the labeling index decreases to 2.8%, and subsequently, the cells begin to acquire the structural characteristics of matured, differentiated endothelium. One-millimeter fragments of fresh Walker 256 carcinoma were implanted on the CAM from day 3 to day 16 [14]. The size of the tumors was measured daily, and the onset of vascularization of each tumor was determined in vivo with a stereomicroscope and confirmed with histological sections. Proliferation of chick capillaries occurred in the neighborhood of the tumor graft by 24 h after implantation, but capillary sprouts did not penetrate the tumor graft until approximately 72 h. During the avascular phase, tumor diameter did not exceed 1 mm. Small tumor implants of 0.5 mm or less grew to 1 mm and stopped expanding. Larger tumor implants of 2- or 3-mm shrank until they reached 1-mm diameter. During the first 24 h after penetration by capillaries, there was a rapid tumor growth. Neovascularization was not grossly observable with the stereomicroscope until after day 10 or 11. Tumors implanted on the CAM after day 11 grow at slower rate in parallel with the reduced rates of endothelial growth.When tumor grafts of increasing size (from 1 to 4 mm) are implanted on the 9-day CAM, grafts larger than 1 mm undergo necrosis and autolysis during the 72-h prevascular phase. They shrink rapidly until the onset of neovascularization, when rapid tumor growth resumes [14]. In another study [17], the behavior of tumor grafts on the CAM was compared to grafts of normal adult and embryonic tissues. In tumor tissue, preexisting blood vessels within the tumor graft disintegrated by 24 h after implantation. Neovascularization did not occur until after at least 3 days, and only by penetration of proliferating host vessels into the tumor tissue. There was marked neovascularization of host vessels in the neighborhood of the tumor graft. By contrast, in embryonic graft, preexisting vessels did not disintegrated. They reattached by anastomosis to the host vessels within 1–2 days, but with minimal or almost no neovascularization on the part of the host vessels. In adult tissues, the preexisting graft vessels disintegrated; there was no reattachment of their circulation with the host, and adult tissues did not stimulate capillary proliferation. These studies suggest that only tumor grafts are capable of stimulating formation of new blood vessels in the host.In transgenic mice that develop carcinomas of the β cells in the pancreatic islets, large tumors arise only from a subset of preneoplastic hyperplastic islets that have become vascularized [18].
Isolation of the first angiogenic tumor factor
Until the early 1970s, it was widely assumed that tumors did not produce specific angiogenic proteins. The conventional widsom was that tumor vasculature was an inflammatory reaction to dying or necrotic tumor cells.
Previous studies had shown that tumor-stimulated vessel growth did not require direct contact between tumor and host tissue [4, 5]. This made sense to Folkman, who reasoned that a soluble factor would be more likely to reach nearly than distant blood vessels. He and his colleagues isolated an angiogenic factor in 1971 [19]. The homogenate of a Walker 256 carcinoma—a breast tumor of Sprague-Dawley rats—was fractionated by gel-filtration on Sephadex G-100. The fraction that exhibited the strongest angiogenic activity had a molecular weight of about 10,000 daltons and consisted of 25% RNA, 10% proteins, and 58% carbohydrates, plus a possible lipid residue. It was inactivated by digestion with pancreatic ribonuclease, or by heating at 56°C for 1 h, and was not modified when kept at 4°C for 3 months, nor when treated with trypsin for more than 3 days. This active fraction was subsequently called “tumor angiogenesis factor” (TAF) [19]. Both the cytoplasmic and the nuclear fractions of tumor cells stimulated angiogenesis. In the nuclear fraction, this was found to be associated with nonhistonic proteins [20]. Tumor angiogenesis factor has since been nondestructively extracted from several tumor cell lines, and several low molecular weight angiogenic factors have been isolated, again from the Walker 256 carcinoma. These factors induced a vasoproliferative response in vivo when tested on rabbit cornea or chick CAM, and in vitro on cultured endothelial cells [21–23].
First evidence of the existence of the avascular and vascular phases of solid tumor growth
The earliest evidence of the existence of the two phases was obtained by Folkman and collaborators in 1963, who perfused the lobe of a thyroid gland with plasma and inoculated a suspension of melanoma B16 tumor cells through the perfusion fluid. These cells grew into small, clearly visible black nodules. The nodules did not exceed 1 mm in diameter and did not connect with the host’s vascular network. Their outer third generally remained vital, while the interior portion underwent necrosis. Reimplanted nodules, on the other hand, equipped themselves with a vascular network and grew very rapidly. The conclusion was thus drawn that the absence of vascularization limits the growth of solid tumors.
Further research by Folkman’s group resulted in an experimental system in which the tumor, or its extracts, could be separated from the vascular bed [24, 25]. This system was based on subcutaneous insufflation to lift the skin of a rat and form a poorly vascularized region below it. Millipore filters containing Walker 256 cancer cells or their cytoplasmic or nuclear extracts (TAF) were implanted into the fascial floor of the dorsal air sac. At intervals thereafter, 3H-labeled thymidine was injected into the air sac, and the tissues were examined by autoradiography and electron microscopy. Autoradiographs showed thymidine-3H labeling in endothelial cells of small vessels, 1–3 mm from the site of implantation, as early as 6–8 h after exposure to tumor cells. DNA synthesis by endothelium subsequently increased, and within 48 h new blood vessels formation was detected. The presence of labeled endothelial nuclei, endothelial mitosis, and regenerating endothelium was confirmed by electron microscopy. Tumor angiogenesis factor also induced neovascularization and endothelial DNA synthesis after 48 h. Further ultrastructural autoradiographic studies were carried out with the same model [25]. It was apparent that by 48 h there was ultrastructural evidence of regenerating endothelium, including marked increase in ribosomes and endoplasmic reticulum, scarce or absent pinocytotic vesicles, and discontinuous basement membrane. Labeled endothelial cells were seen along newly formed sprouts as well as in parent vessels. Furthermore, pericytes were also shown to synthesize DNA.
In another series of experiments, 1-mm fragments from Brown-Pearce and V2 carcinomas were implanted into the avascular stroma of a rabbit cornea 1–6 mm away from the limbic vessels, and observed the tumor growth daily with a stereomicroscope [10]. After 1 week, new blood vessels had invaded the cornea starting from the edge closer to the site of implantation and developed in that direction at 0.2 mm and then about 1 mm/day. Once the vessels reached the tumor, it grew very rapidly to permeate the entire globe within 4 weeks.
Dormancy of micrometastases may be governed by angiogenesis
Folkman and collaborators found that metastases were suppressed when a primary tumor was implanted and allowed to grow in nude mice, whereas they underwent neovascularization and became clinically evident when primary neoplasm was removed. In the absence of angiogenesis, micrometastases rarely exceeded 0.2-mm diameter and contained many proliferating tumor cells balanced by many apoptotic cells. When they were allowed to become angiogenic, they grew rapidly. Dormancy may be generalizable to a variety of tumors in which blocked angiogenesis results in balanced tumor cell proliferation and apoptosis [26].
Prognostic significance of tumor vascularity
In 1972, Brem in the Folkman laboratory reported the first quantitative method for histologic grading of tumor angiogenesis. He correlated neovascularization in human brain tumors with tumor grade [27]. In the early 1990s, Weidner and collaborators [28–30] showed that measurement of microvascular density within isolated regions of high vessel concentration (i.e., hotspots) was a prognostic indicator for human breast and prostate carcinomas.
Microvascular density counting protocols have become the morphological gold standard to assess the neovasculature in human tumors. This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microvessels. Microvascular density determined in primary tumors is significantly associated with metastasis and prognosis in several solid and hematological tumors.
Antiangiogenesis
The existence of specific angiogenesis inhibitors was first postulated by Folkman in 1971 in an editorial. No angiogenesis inhibitors existed before 1980, and few scientists thought at that time that such molecules would ever be found.
From 1980 to 2005, Folkman’s laboratory reported the discovery of 12 angiogenesis inhibitors (Table 1). The first angiogenesis inhibitor was found in cartilage, an avascular tissue that resists invasion by many tumors [31]. Brem and Folkman demonstrated that tumor-induced vessels were inhibited by a diffusible factor from neonatal rabbit cartilage [32]. The partially purified inhibitor suppressed tumor growth when it was infused into the vascular bed of murine and rabbit tumors [33]. 2-methoxyestradiol was first reported by Fotsis et al. [34], and the article reporting 2-methoxyoestradiol and its molecular mechanism as an inhibitor of tubulin polymerization by acting at the colchicine site was published a month later [35].
Table 1Angiogenesis inhibitors discovered in Folkman’s laboratory from 1980 to 20051980. Interferon alpha-beta1982. Platelet factor 4/protamine1985. Angiostatic steroids1990. Fumagillin1994. Angiostatin1994. Thalidomide1994. 2-methoxyestradiol1997. Endostatin1999. Cleaved antithrombin III2002. 3-Aminotholidomide2003. DBF-maf2005. Caplostatin
Interferon alpha
Interferon alpha was first shown to inhibit endothelial cell migration in a dose-dependent and reversible manner in 1980 by Zetter in Folkman’s laboratory [36]. Since 1988, interferon alpha has been used successfully to cause complete and durable regression of life-threatening pulmonary hemangiomatosis, hemangiomatosis of the brain, airway and liver in infants, recurrent high-grade giant cell tumors refractory to conventional therapy, and angioblastomas [37–39].
A 12-year-old boy with fatal pulmonary hemangiomatosis had a complete remission and recovered completely after 7 months of therapy with interferon alpha. Therapy was continued for 7 years. This led to the successful use of low-dose daily interferon alpha therapy administered subcutaneously to infants with sight-threatening or life-threatening hemangiomas and hemangioendotheliomas of the heart, airway, and liver [37–39].
Platelet factor 4/protamine
Protamine was shown to be an angiogenesis inhibitor [40], but cumulative toxicity from prolonged administration and a narrow window of angiostatic efficacy prevented its consideration for clinical use.
Platelet factor 4 was first tested for antiangiogenic activity because its method of binding and neutralizing heparin is similar to that of protamine [40]. Recombinant human platelet factor 4 (rHuPF4) has been produced [41]. It specifically inhibited endothelial proliferation and migration in vitro [42]. The inhibitory activities are associated with the carboxy-terminal region of the molecule. The growth of human colon carcinoma in athymic mice, as well as the growth of murine melanoma, was markedly inhibited by intralesional injections, whereas tumor cells were completely insensitive to rHuPF4 in vitro at levels that inhibited normal endothelial cell proliferation. Systemic administration of rHuPF4 has so far been ineffective against tumor growth, perhaps because of rapid inactivation or clearance of the peptide.
Angiostatic steroids
Folkman had begun to use the CAM of the chick embryo to detect angiogenic activity in fractions being purified from tumor extracts. The addition of heparin increased the speed of development of the angiogenic reaction so that it could be read 1–2 days later [40]. But one problem with this assay is that occasionally eggshell dust falls on the CAM and causes background inflammation. Folkman guessed that adding cortisone to the CAM might eliminate the irritation from the shell dust but not abolish the tumor-angiogenic reaction. As expected, cortisone alone prevented shell dust inflammation without interfering with angiogenesis induced by tumor extracts. The surprise was that when heparin and cortisone were added together, tumor angiogenesis was inhibited [43]. Furthermore, when this combination of heparin and steroid was suspended in a methylcellulose disk and implanted on the young (6-day) CAM, growing capillaries regressed leaving in their place, 48 h later, an avascular zone. The antiangiogenic effect was specific for growing capillaries. Mature nongrowing capillaries in older membranes were unaffected. Nonanticoagulant heparin had the same effect. A hexasaccharide fragment with a molecular weight of approximately 16,000 was found to be the most potent inhibitor of angiogenesis (in the presence of a corticosteroid). The combination of the heparin hexasaccharide fragment and cortisone also inhibited tumor-induced angiogenesis in the rabbit cornea.
The regression of a growing vessel exposed to heparin-steroid combinations begins with endothelial cell rounding and is followed by cessation of endothelial proliferation, desquamation of endothelial cells, and retraction of the capillary sprout [44]. These events occur as 24–48 h and are accompanied by dissolution of the basement membrane of the new capillary vessels.
Fumagillin
Fumagillin was found by Ingber in the Folkman laboratory to inhibit endothelial cell proliferation without causing endothelial cell apoptosis, when a tissue culture plate of endothelial cells became contaminated with a fungus Aspergillus fumigatus fresenius [45]. A conditioned medium from fungal cultures contained an inhibitor of endothelial cell proliferation and angiogenesis, which, upon purification, was found to be fumagillin, a polyene macrolide. When capillary endothelial cells were stimulated by fibroblast growth factor-2 (FGF-2), half-maximal inhibition was observed with fumagillin at 100 pg/ml. This antiproliferative effect appeared to be relatively specific for endothelial cells because inhibition of nonendothelial cells, including tumor cells, was observed at up to 1,000-fold higher concentrations.
Scientists at Takeda Chemical Industries (Osaka, Japan) made a synthetic analogue of fumagillin, called TNP-470, which inhibits endothelial proliferation in vitro at a concentration 3 logs lower than the concentration necessary to inhibit fibroblasts, and tumor cells.
Angiostatin and endostatin
They were discovered by M. O’Reilly in Folkman laboratory based on Folkman’s hypothesis of a mechanism to explain the phenomenon that surgical removal of certain tumors leads to rapid growth of remote metastases. This hypothesis said that if tumors produce both stimulators and inhibitors of angiogenesis, an excess of inhibitors could accumulate within an angiogenic tumor. In the circulation however, the ratio would be reversed. Angiogenesis inhibitors would increase relative to stimulators, because of rapid clearance of stimulators from the blood. Folkman formulated this hypothesis after reading Bouck’s first report in 1989 that the emergence of tumor angiogenesis was the result of a shift in balance between positive and negative regulators of angiogenesis in a tumor [46]. Bouck reported that the switch to angiogenesis during tumorigenesis of transformed hamster cells was associated with downregulation of an inhibitor of angiogenesis, thrombospondin. She suggested that the switch to the angiogenic phenotype could be the result of a shift in the net balance of positive and negative regulators of angiogenesis.
In 1991, O’Reilly began to screen a variety of transplantable murine tumors for their ability to suppress metastases. A Lewis lung carcinoma was the most efficient. When the metastasis-suppressing primary tumor was present in the dorsal subcutaneous position, microscopic lung metastases remained dormant at a diameter of less than 200 μm surrounding a preexisting microvessel, but revealed no new vessels. Within 5 days after surgical removal of the primary tumor, lung metastases became highly angiogenic and grew rapidly, killing their host by 15 days [47]. This striking evidence that primary tumor could suppress angiogenesis in its secondary metastases by a circulating inhibitor was further supported by the demonstration that a primary tumor could also suppress corneal angiogenesis by an implanted pellet of FGF-2. O’Reilly then succeeded in purifying this inhibitor from the serum and urine of tumor-bearing animals. It was a 38-kD internal fragment identical in amino acid sequence to the first four kringle structures of plasminogen and it was named angiostatin. Angiostatin specifically inhibited the proliferation of growing vascular endothelial cells and had no effect on resting confluent endothelial cells or on other cell types, including smooth muscle cells, epithelial cells, fibroblasts and tumor cells. It also inhibited growth of primary tumors by up to 98% [48] and was able to induce regression of large tumors (1–2% of body weight) and maintain them at a microscopic dormant size.
Based on the same rationale and strategy, O’Reilly isolated and purified another angiogenesis inhibitor from a murine hemangioendothelioma. This inhibitor, called endostatin [49], is a 20-kD protein with an N-terminal amino acid sequence identical to the carboxyterminus of collagen XVIII. It was purified directly from tumor cell-conditioned medium. Endostatin is also a specific inhibitor of endothelial proliferation and has no effects on resting endothelial cells or on other cell types. It is slightly more potent than angiostatin and also causes regression of large tumors to a microscopic size.
Thalidomide
In 1994, D’Amato in the Folkman laboratory reported that thalidomide is an angiogenic inhibitor [50]. Corneal neovascularization in rabbits induced by FGF-2 or VEGF was blocked by orally administered thalidomide. This activity of thalidomide was mainly the result of its direct effect on inhibiting new blood vessel formation and not on suppression of infiltrating host inflammatory cells. Histologic sections of the pretreated neovascularized corneas were virtually free of inflammatory cells. Thalidomide also inhibited corneal neovascularization in mice, but it was necessary to give the drug by the intraperitoneal route and at high doses, because mice do not metabolize thalidomide effectively.
2-Methoxyestradiol
In 1994, D’Amato in Folkman laboratory demonstrated that a metabolite of estradiol, 2-methoxyestradiol, inhibited angiogenesis in the chick CAM [35]. Moreover, since 2-methoxyestradiol causes mitotic perturbations, they examined its interactions with tubulin and showed that 2-methoxyestradiol bound to colchicine site of tubulin and, depending on reaction conditions, either inhibited assembly or seems to be incorporated into a polymer with altered stability properties.
Cleaved antithrombin III
A human small-cell lung carcinoma suppressed angiogenesis and tumor growth at remote sites in immunodeficient mice. These cells generated an enzyme in vitro that converted the 58-kD conformation of circulating antithrombin III to a 53-kD form of the protein [51] in which the externally configured stressed loop of antithrombin was retracted into the body of the molecule. The 53-kD form is a specific endothelial inhibitor and a potent angiogenesis inhibitor and has no thrombin binding activity.
Caplostatin
Caplostatin is a nontoxic synthetic analogue of fumagillin conjugated to a water-soluble-N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer [52, 53]. Caplostatin has a similar broad antitumor spectrum of TNP-470 without any toxicity. In addition to its antiangiogenic activity, caplostatin is the most potent known inhibitor of vascular permeability [53]. Caplostatin prevents vascular leakage induced by VEGF, bradykinin, histamine, and platelet-activating factor and prevents pulmonary edema induced by interleukin-2.
Antiangiogenic chemotherapy
Browder in the Folkman laboratory was the first to demonstrate the concept that by optimizing the dosing schedule of conventional cytotoxic chemotherapy to achieve more sustained apoptosis of endothelial cells in the vascular bed of a tumor, it is possible to achieve more effective control of tumor growth in mice, even if the tumor cells are drug resistant [54]. Conventional chemotherapy is administered at maximum tolerated doses followed by off-therapy intervals of 2–3 weeks to allow the bone marrow and gastrointestinal tract to recover. In contrast, antiangiogenic chemotherapy is administered more frequently at lower doses, without long interruptions in therapy and with little or no toxicity. During antiangiogenic chemotherapy, endothelial cell apoptosis and capillary dropout precede the death of tumor cells that surround each capillary.
Concluding remarks
Currently, several compounds with angiostatic activity are approved for clinical use, and many are in late-stage clinical development. However, the results from clinical trials have not shown the antitumor effects which were expected following preclinical studies. It appears that clinical applications of antiangiogenic therapy are more complex than originally thought.
The main problem in the development of antiangiogenic agents is that multiple angiogenic molecules may be produced by tumors, and tumors at different stages of development may depend on different angiogenic factors for their blood supply. Therefore, blocking a single angiogenic molecule was expected to have little or no impact on tumor growth. Current development of targeted antiangiogenic agents include their use in adjuvant settings and the combination of different antiangiogenic inhibitors to take a more comprehensive approach in blocking tumor angiogenesis.
Advancing insights into fundamental mechanisms will be necessary in the development of novel anticancer strategies based on inhibition of angiogenesis.

angiogenesis
antiangiogenesis
history of medicine
tumor progression


Immunohistochemical detection of connexin36 in sympathetic preganglionic and somatic motoneurons in the adult rat

Gap junctional communication in the adult CNS plays an important role in the synchronization of neuronal activities. In vitro studies have shown evidence of electrotonic coupling through gap junctions between sympathetic preganglionic motoneurons and between somatic motoneurons in the neonatal and adult rat spinal cord. Electrotonic transmission of membrane oscillations might be an important mechanism for recruitment of neurons and result in the generation of rhythmic sympathetic and somato-motor activity at the population level. Gap junctions in the adult spinal cord are constituted principally by connexin36 (Cx36). However, the distribution of Cx36 in specific neuronal populations of the spinal cord is unknown. Here, we identify Cx36-like immunoreactivity in sympathetic preganglionic and somatic motoneurons in thoracic spinal cord segments of the adult rat. For this purpose, double immunostaining against Cx36 and choline acetyltransferase (ChAT) was performed on transverse sections (20 μm) taken from spinal segments T6–T8. Cx36 punctate immunostaining was detected in the majority of ChAT-immunoreactive (-ir) neurons from lamina VII [intermediolateral cell column (IML) and intercalated cell group (IC)], lamina X [central autonomic nucleus (CA)] and in ventral horn neurons from laminae VIII and IX. Cx36 puncta were distributed in the neuronal somata and along dendritic processes. The presence of Cx36 in ChAT-ir neurons is consistent with electrical coupling between sympathetic preganglionic motoneurons and between somatic motoneurons through gap junctions in the adult spinal cord.
1
Introduction
Gap junctions are specialized cell–cell contacts that provide direct intercellular communication. They often occur as plaques that can contain thousands of individual gap junction channels. Each channel is formed by the association of 12 connexin (Cx) proteins. Eleven of the 20 members connexin family have been identified so far in the mammalian central nervous system (Nagy et al., 2004), but only Cx36 is expressed almost exclusively by neural cells (Belluardo et al., 2000; Condorelli et al., 2000; Rash et al., 2000). Previous studies have shown that gap junctions play an important role in the synchronization of neuronal activity in various areas of the neonatal and adult rodent brain, including the cortex (Deans et al., 2001), hippocampus (Hormuzdi et al., 2001; Buhl et al., 2003), thalamic reticular nucleus (Landisman et al., 2002), inferior olive (Long et al., 2002; De Zeeuw et al., 2003; Leznik and Llinas, 2005) and olfactory bulb glomeruli (Christie et al., 2005). Such junctions enable the intercellular flow of electric current (electrotonic transmission) which can result in the generation of coordinated activity within populations of neurons. In the rat spinal cord, electrotonic and dye coupling have been shown to occur between somatic motoneurons (Gogan et al., 1977; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002) and between sympathetic preganglionic motoneurons (Logan et al., 1996; Nolan et al., 1999). It has been suggested that synchronization of electrical activity through gap junctions plays an important role in the generation of somatomotor and sympathetic motor rhythms induced by NMDA and/or 5-HT (Marina et al., 2006; Tresch and Kiehn, 2000). In the spinal cord of adult rats, gap junctions are composed mainly by Cx36 and are distributed in the white matter and the grey matter (Rash et al., 2000; Lee et al., 2005). However, the expression of Cx36 protein in specific neural populations of the spinal cord remains unknown. In this study we used immunofluorescence to identify Cx36-like immunoreactivity in ChAT-positive putative sympathetic preganglionic and somatic motoneurons within thoracic spinal segments.
2
Materials and methods
2.1
Tissue preparation
Experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. Six male Sprague–Dawley rats (210–250 g, bred in-house at UCL) were anaesthetized deeply with 20% urethane (1.6 g kg− 1 i.p.) and perfused through the ascending aorta with 100 ml of 0.9% saline at room temperature, followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.4) at 4 °C. The thoracic spinal cord and the brain stem were removed, postfixed for 1 h and cryoprotected in 10% sucrose in 0.1 M PBS overnight at 4 °C. Transverse slices (20 μm thick) from spinal segments T6–T8 and caudal medulla were collected with a freezing microtome and stored in cryo-protectant (30% ethylene glycol and 20% glycerol in 0.05 M PBS) at −20 °C.
Caudal medulla slices were taken to assay specificity of the Cx36 antibody and to determine optimal antibody concentration using the inferior olive nucleus (ION) as a positive control. Cx36-immunostaining was analyzed in spinal segments T6–T8 because previous studies have shown evidence of electrotonic coupling among sympathetic preganglionic motoneurons and expression of Cx36 mRNA in somatic motoneurons from thoracic spinal cord slices (Logan et al., 1996; Parenti et al., 2000).
2.2
Immunohistochemistry
All series of tissue slices from all animals (20–30 slices/animal) were processed together in the same assay to ensure uniformity of immunostaining. After slices were washed in 0.1 M PBS with 0.1% Triton X (PBS-T), non-specific binding was blocked with 10% normal donkey serum (Sigma-Aldrich) in PBS-T (blocking medium) for 60 min. Tissue was then incubated in Cx36 antibody (1:100 dilution in blocking medium) at 4 °C overnight. The antibody was obtained by immunizing rabbits with a synthetic peptide corresponding to amino acids LQNTETTSKETEPDC of the murine Cx36 protein. The peptide sequence was compared to protein sequence databases from the Basic Local Alignment Search Tool (BLAST 2.0, NCBI, National Institutes of Health, USA), and showed local alignments with murine Cx36 protein only. The specificity of the Cx36 antibody was shown in Western blots and in preabsorption control experiments (Bittman et al., 2002). The slices were then washed again with PBS-T and incubated with fluorescent secondary antibody (Alexa Fluor 488 donkey anti-rabbit IgG, Molecular Probes, 1:500 dilution in blocking medium) for 60 min at room temperature, and washed again with PBS.
Choline acetyltransferase (ChAT) immunodetection was employed to identify putative sympathetic preganglionic and somatic motoneurons (Marina et al., 2002). Following staining for Cx36, slices were incubated in goat anti-ChAT affinity purified polyclonal antibody (Chemicon, 1:100 dilution in 1% normal donkey serum) overnight at 4 °C. Tissue was washed and incubated with Alexa Fluor 568 donkey anti-goat IgG antibody (Molecular Probes, 1:500 dilution in 1% normal donkey serum) for 60 min at room temperature, washed again in PBS and cover-slipped with Vectashield hard set mounting medium with the fluorescent nuclear dye DAPI (Agar Scientific, Stansted, Essex).
2.3
Image analysis
The anatomical distribution of Cx36 immunostaining was determined by visual inspection through 63× and 100× objectives with an Olympus BH2-RFCA epifluorescence microscope equipped with light filters (exciter 380–490 nm, emitter 519 nm for the green channel). ChAT-Immunoreactive (ir) neurons were considered positive for Cx36 if punctate staining was observed within the cell body or along the dendritic processes.
The cellular distribution of Cx36 in sympathetic preganglionic and somatic motoneurons was analyzed using a Leica TCS NT SP laser scanning confocal microscope with argon, krypton, and helium-neon lasers. Green and red channels were each scanned sequentially through a 63× objective. Alexa Fluor 488 labelling was observed by exciting at 488 nm and collecting at 515–550 nm. Alexa Fluor 568 labelling was visualized by using excitation at 568 nm and collecting at 590–626 nm. Serial optical sections were taken at intervals of 0.5 μm starting at the upper cell surface, i.e., the first optical section where cytoplasmic ChAT staining became visible, and all the way down through the cell to the lower cell surface, i.e., where ChAT staining was no longer detected. Composite figures were created for each optical section by merging both channels using Image J 1.37c (National Institutes of Health, USA). Colocalization of Cx36 (green) and cytoplasmic ChAT immunofluorescence (red) appeared as yellow. Thus, green puncta on the cell surface, i.e., surrounding ChAT-ir cytoplasm, suggested the presence of Cx36 on the plasma membrane; yellow intracellular puncta in ChAT-positive regions were indicative of cytoplasmic Cx36. Serial sections were projected as a single image to create a 2D reconstruction of 3D data set from the sections of the cell bodies and dendritic processes, using Leica Confocal Software (Leica Microsystems Heidelberg GmbH). Both the individual and projected images were saved directly to a computer as TIFF files. Images were minimally adjusted for brightness and contrast with Adobe Photoshop® 6 running on a Dell Pentium 4 PC. Images were then imported into Adobe illustrator® CS2 where groups of images were assembled and labelled.
3
Results
3.1
Connexin 36 immunostaining in the inferior olivary nucleus
Caudal medulla slices were immunostained for Cx36 to be used as a positive control. Cx36-immunostaining could only be detected using high magnification lenses (at least 63×) and appeared as minuscule round immunofluorescent puncta distributed in restricted regions within the inferior olivary nucleus (Fig. 1A). Cx36 expression in other medullary nuclei was extremely low, as illustrated in Fig. 1B, where Cx36-like-ir puncta are virtually undetectable in the trigeminal spinal nucleus. No staining was observed in negative controls where the primary antibody was omitted (data not shown).
3.2
Connexin36 immunostaining in the spinal cord
Under low magnification (40× or less), Cx36 immunostaining is very hard to detect as immunofluorescent puncta are small and sparsely distributed in the ventral horn (Rash et al., 2000; Lee et al., 2005). Under high magnification (63× and higher), several Cx36-like-ir puncta were revealed within the soma of spinal neurons (Fig. 2).
3.3
Choline acetyltransferase immunostaining in the spinal cord
Sympathetic preganglionic and somatic motoneurons were identified by immunohistochemical staining of ChAT in transverse spinal cord slices from thoracic spinal segments T6–T8. Intracellular ChAT immunostaining (red immunofluorescence) was detected extranuclearly in the cytoplasm and in the dendritic projections (Figs. 2 and 3). ChAT-ir neurons located in lamina VII (IML and IC) and lamina X (CA) were considered as sympathetic preganglionic motoneurons, according to the spatial arrangement described by Cabot (1990; see Fig. 2A). Lateral and medial ChAT-ir projections were easily identified in all sympathetic preganglionic motoneurons within the IML; dorsal and ventral dendritic projections were not identified in transverse spinal slices stained for ChAT. On the other hand, ChAT-ir neurons and their respective dendritic projections located in laminae VIII and IX were considered as somatic motoneurons, according to the anatomical boundaries described by Paxinos and Watson (1998; see Fig. 3).
3.4
Connexin 36 immunostaining in sympathetic preganglionic motoneurons
The presence of Cx36-like-ir puncta in transverse spinal cord slices taken from T6–T8 spinal segments immunostained for ChAT was analyzed with wide field epifluorescence microscopy. The cellular distribution of Cx36-like-ir puncta in putative sympathetic preganglionic and somatic motoneurons was analyzed in individual optical sections scanned sequentially along the cell body (z series) under high magnification. Optical sections were stacked to show a three-dimensional reconstruction of the neurons. Figs. 2 and 3 show examples of somal and dendritic labelling in ChAT-ir neurons taken from a representative spinal slice from animal number 5. Co-expression of Cx36 (green) and ChAT (red) immunofluorescence appears as yellow. Cx36 immunostaining in ChAT-ir neurons was similar in all the slices taken from all six animals used in this study. All ChAT-ir neurons located in the different sympathetic nuclei, i.e., IML, IC and CA, showed Cx36-like-ir puncta (See Fig. 2A and A1). Both, somal and dendritic puncta were observed in putative sympathetic preganglionic motoneurons. A detailed analysis of each optical section revealed that membrane labelling was very scarce; this was indicated by the small amount of green puncta surrounding ChAT-ir cytoplasm (Figs. 2A1). In contrast, moderate intracellular punctate staining was detected in ChAT-ir regions, i.e., perinuclearly, where the Cx36 is generated in the Golgi-ER complex, suggesting a substantial cytoplasmic production of Cx36 protein. On the other hand, Cx36 staining was consistently detected in ChAT-ir dendrites projected from sympathetic preganglionic neurons within the IML, i.e., lateral and medial projections. Dendritic labelling was characterized by fine puncta distributed along the primary dendrites and thin filaments. Robust Cx36-like staining was often observed in medially projecting bundled dendrites (Fig. 2B1). In some cases, fine puncta were also detected in areas where fine dendrites were seen in close proximity to a cell body (Fig. 2B1). As mentioned before, dorsal dendritic projections were not detected in transverse spinal slices stained for ChAT, and therefore, the presence of Cx36-like-ir puncta in dorsal dendrites could not be determined.
3.5
Connexin 36 immunostaining in somatic motoneurons
Cx36-immunoreactive puncta were consistently observed in somatic motoneurons from laminae VIII and IX. Cx36 labelling in somatic motoneurons was characterized by moderate punctate staining distributed perinuclearly in the cytoplasm and to a lesser extent on the plasma membrane. Punctate staining was also distributed along the dendritic arbour and it was often observed in dendro-dendritic and somato-dendritic sites of close apposition (Fig. 3).
4
Discussion
4.1
Methodological considerations
In the present study, connexin 36-immunoreactivity was identified in putative sympathetic preganglionic motoneurons and somatic motoneurons from the lower thoracic spinal cord of adult rats. The staining pattern obtained was similar to that described previously in the spinal cord and in the ION (Rash et al., 2000; Placantonakis et al., 2006). Even though the peptide used to immunize the rabbits showed local alignments with murine Cx36 protein only, it is important to mention that the specificity of the antibody has only been partially tested in preabsorption control experiments and in Western blots (Bittman et al., 2002). Further experimental evidence is required to fully determine the specificity of this antibody using tissue from Cx36 knock-out animals.
It is important to consider that connexin expression can only be estimated at the subcellular level using high magnification objectives. Cx 36-ir puncta are quite small, with an average diameter of 0.61 μ (Placantonakis et al., 2006) and Cx36 protein levels in the adult spinal cord are distinctly low. Therefore, visualization at low magnification can be extremely difficult; this point can also be illustrated by the apparent difference in the expression of Cx36mRNA levels in the adult spinal cord described in previous studies. Showing a panoramic photograph of the spinal cord at low magnification (judged by the scale bars), Lee et al. (2005) showed that Cx36 mRNA is strong in the neonatal rat spinal cord but in the adult spinal cord, expression is very low and barely above background. In contrast, Cx36 mRNA expression in the adult rat spinal cord can be clearly seen in higher magnification images by Chang et al. (2000). In the present study, it was also difficult to identify Cx36-like-ir puncta in panoramic images of the spinal cord (see Fig. 1A). However, a closer inspection revealed the presence of Cx36 within ChAT-ir neurons.
4.2
Cellular distribution of Cx36 immunostaining in ChAT-ir neurons
This study shows that ChAT-ir neurons from the adult rat thoracic spinal cord express the neuron-specific gap junction protein Cx36. Such labelling was detected both in the IML, IC and CA and in laminae VIII and IX and it was distributed in neuronal somata and along dendritic processes. Somal labelling was mainly characterized by moderate cytoplasmic punctate staining with scarce labelling on the plasma membrane and cell nucleus. Like all membrane proteins, connexins are synthesized by ribosomes and are transported in small vesicles that follow the cell's secretory pathway from the endoplasmic reticulum to the plasma membrane (Yeager et al., 1998; Martin and Evans, 2004). Removal of gap junctions occurs through the formation of annular gap junctions by phagocytosis and lysosomal degradation (Yeager et al., 1998). Therefore, the cytoplasmic labelling observed in putative sympathetic preganglionic motoneurons and in somatic motoneurons may represent the trafficking pathways of connexins leading to the formation and/or degradation of gap junction channels. Our results therefore support the contention that Cx36 is actively synthesized by the adult spinal cord. This is the first documentation of connexin 36-like immunostaining in ChAT-ir (putative sympathetic preganglionic) neurons in sympathetic nuclei of the spinal cord. Furthermore, our results are consistent with several studies, which have demonstrated that Cx36 mRNA is widely expressed in somatic motoneurons during the embryonic and neonatal life, and continues to be expressed in the adulthood (Chang et al., 1999; Chang et al., 2000; Parenti et al., 2000).
Cx36 labelling was scarce on the somatic plasma membrane of ChAT-ir neurons and it was never found in sites of close apposition between neighbouring cell bodies. However, abundant Cx36 labelling was identified in and along dendritic processes. In fact, Cx36-like-ir puncta were commonly detected in tightly bundled dendrites and in areas where fine dendrites were in close proximity with other cell bodies. The presence of Cx36 in these areas might represent a potential site for gap junctional coupling between dendrites and between the somata and the dendrites of the neighbouring sympathetic preganglionic and somatic motoneurons. This is consistent with in vitro studies using slice preparations, which have identified putative sites of coupling between dendrites and/or cell bodies in dye-coupled sympathetic preganglionic and somatic motoneurons (Becker and Navarrete, 1990; Shen and Dun, 1990; Mentis et al., 2002). This has also been confirmed at the ultrastructural level, where dendro-dendritic gap junctions have been identified in somatic motoneurons (van der Want et al., 1998). In fact, gap junctions in somatic motoneurons are distributed predominantly in dendro-dendritic and dendro-somatic sites and to a much lesser extent, in somato-somatic points (Matsumoto et al., 1988). Further ultrastructural studies are required to determine whether dendritic Cx36 protein is involved in the formation of homotypic gap junctional plaques among sympathetic preganglionic motoneurons or even heterotypic gap junctions between sympathetic preganglionic motoneurons and interneurons or astrocytes. For instance, previous studies have shown the presence of heterotypic gap junctions in the lumbar spinal cord, composed of Cx32 in superficial dorsal horn neurons on one side, and Cx43 in astrocytic processes on the other (Qin et al., 2005). Interestingly, neuronal tracing studies have proposed the presence of dendro-dendritic connections (presumably through gap junctions) between lamina V neurons from the dorsal horn and sympathetic preganglionic motoneurons from the IML (Cabot et al., 1994). It will be interesting to determine whether Cx36 in sympathetic preganglionic motoneurons is involved in the formation of heterotypic gap junctions with lamina V neurons.
4.3
Physiological role of gap junctional communication between sympathetic preganglionic motoneurons
Paired cell recordings have shown evidence of synchronization of action potential firing and subthreshold membrane potential oscillations between electrotonically coupled sympathetic preganglionic motoneurons (Logan et al., 1996; Nolan et al., 1999). Since action potential discharge in one of the sympathetic preganglionic motoneurons was almost simultaneously registered as a spikelet in the second sympathetic preganglionic neuron, it was considered that gap junctions mediating electrotonic coupling are located near the site of action potential generation (Logan et al., 1996). These data are consistent with the abundance of the gap junction protein Cx36 that we found between primary dendrites in sympathetic preganglionic motoneurons and supports the idea that electrical synapses might be located in dendro-dendritic sites of close apposition. Electrical synapses in sympathetic preganglionic motoneurons behave in a similar way to a low-pass filter, allowing electrotonic transmission of sub- and suprathreshold activity (Nolan et al., 1999). Therefore, it has been recently proposed that by increasing the degree of synchrony of discharges in the population of sympathetic preganglionic motoneurons, gap junctional communication might play a significant role in the generation of relatively low frequency coherent sympathetic motor rhythms induced by 5-HT in the adult rat (Marina et al., 2006).
4.4
Physiological role of gap junctional communication between somatic motoneurons
Several studies have shown evidence of electrical and dye coupling between somatic motoneurons in the neonatal spinal cord (Gogan et al., 1977; Becker and Navarrete, 1990; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002). Experiments using gap junction blockers and NMDA receptor antagonists have shown that coherent somato-motor rhythms result from the interaction between membrane properties at the single cell level and the transmission of this activity through gap junctions (see Kiehn and Tresch, 2002). It has been suggested that gap junction coupling is a very important factor that contributes to the synchronization of individual somatic motoneurons to produce a coordinated rhythmic motor output.
The dye transfer properties between neonatal somatic motoneurons change as development progresses, moving from Lucifer yellow to neurobiotin coupling (Becker and Navarrete 1990; Mentis et al., 2002). The number and intensity of labelling of dye-coupled somatic motoneurons are substantially reduced and becomes almost absent shortly after birth (Gogan et al., 1977; Becker and Navarrete, 1990; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002). Electrotonic coupling between somatic motoneurons in adult animals has only been observed in pathological conditions such as nerve injury (Chang et al., 1999). However, Cx36 mRNA expression in axotomized somatic motoneurons remains unaffected (Chang et al., 1999). In a similar way, recent evidence has shown that spinal cord injury is associated with a strong up-regulation of Cx43 in astrocytes, but in contrast, neural Cx36 expression remains unaffected (Lee et al., 2005). This suggests that an increased synthesis of Cx36 protein is not involved in the reappearance of coupling in adult injured animals (Chang et al., 1999). Therefore, it might be possible that the reappearance of coupling between adult somatic motoneurons following injury might be produced by different mechanisms, such as the activation of gap junctions that contain different connexins other than Cx36 or by the release of factors that affect either connexin assembly into functional gap junctions, or the open state of the channels. In fact, several studies have shown that gap junction coupling can be modified by the activation of multiple G protein-coupled receptors, by pH and intracellular calcium levels (see Bennett, 1997; Bennett and Zukin 2004). For instance, early postnatal blockade of NMDA receptors has been shown to prevent the decrease in electrotonic and dye coupling observed during the first postnatal week (Mentis et al., 2002). These data show the importance of gap junction coupling regulation by neuromodulators and suggest the possibility that electrotonic coupling between somatic motoneurons could also reappear in the adult animal under different kinds of pathological states, or even in physiological conditions. To date, this possibility has not been explored.
5
Conclusions
Our results confirm and expand previous observations regarding Cx36 expression in the spinal cord (Rash et al., 2000; Lee et al., 2005) and provide anatomical evidence that suggests that gap junctions composed by Cx36 may mediate electrotonic coupling between sympathetic preganglionic motoneurons and between somatic motoneurons. Further work using different approaches, such as blockers for Cx36 gap junction channels, Cx36-knock-out animals, transfection of sympathetic preganglionic motoneurons and somatic motoneurons with dominant-negative constructs of Cx36 and Cx36-specific antisense oligodeoxynucleotides, will reveal the precise role of Cx36 in the generation of coherent sympathetic and somato-motor rhythmic activity in the spinal cord of adult animals.

sympathetic
motoneurons
gap junctions
spinal cord
rhythmic