Structure and organization of phycobilisomes on membranes of the red alga Porphyridium cruentum

In the present work, electron microscopy and single particle averaging was performed to investigate the supramolecular architecture of hemiellipsoidal phycobilisomes from the unicellular red alga Porphyridium cruentum. The dimensions were measured as 60 × 41 × 34 nm (length × width × height) for randomly ordered phycobilisomes, seen under high-light conditions. The hemiellipsoidal phycobilisomes were found to have a relatively flexible conformation. In closely packed semi-crystalline arrays, observed under low-light conditions, the width is reduced to 31 or 35 nm, about twice the width of the phycobilisome of the cyanobacterium Synechocystis sp. PCC 6803. Since the latter size matches the width of dimeric PSII, we suggest that one PBS lines up with one PSII dimer in cyanobacteria. In red algae, a similar 1:1 ratio under low-light conditions may indicate that the red algal phycobilisome is enlarged by a membrane-bound peripheral antenna which is absent in cyanobacteria.
Introduction
In cyanobacteria and red algae, the main accessory light-harvesting complexes are the phycobilisomes (PBSs), large and highly structured assemblies of phycobiliproteins associated to the cytoplasmic surface of the thylakoid membrane (Gantt 1980; Grossman et al. 1993; MacColl 1998; Adir 2005). They capture and transfer energy primarily to chlorophyll (Chl) a in Photosystem (PS) II (Gantt 1986; Grossman et al. 1993, 1995; Sidler 1994; MacColl 1998; Samsonoff and MacColl 2001; Adir 2005). The PBSs are composed of α- and β- polypeptides bearing covalently attached open-chain tetrapyrroles (Betz 1997). They assemble in disc-shaped aggregates that are stacked in the peripheral rods of the PBS. Colourless linker polypeptides between adjacent discs stabilize the PBS structure and regulate energy transfer (Liu et al. 2005).
PBSs in the unicellular red alga Porphyridium cruentum were first observed about 40 years ago (Gantt and Conti 1966) and described as hemiellipsoidal. Based on electron microscopic results, a preliminary model was proposed (Gantt et al. 1976) involving a tricylindrical core subassembly in the semi-spherical centre and several peripheral rods radiating out from the centre to form the rounded surface of the semi-sphere. The core contains allophycocyanin (APC), while the peripheral rods are mainly composed of phycocyanins (PC) and phycoerythrins (PE). In other systems, hemidiscoidal PBSs have been identified of which the 3-D structure was recently characterized in some detail by single-particle electron microscopy (Barber et al. 2003; Yi et al. 2005). However, such detailed information of hemiellipsoidal PBSs is still lacking.
Despite the fact that energy transfer and mutational studies have indicated that phycobilisomes can couple directly to PSI as well as to PSII, little is known about the structural aspects of the interaction. The precise association of phycobilisomes to PSII and PSI is not known, but there are some hints which PSII subunits could be involved in interaction. A structural comparison between the core parts of green plant and cyanobacterial PSII shows differences in the position of the cyt b559 subcomplex and of the CP47 core antenna with respect to the D1/D2 reaction centre. Within the D1/D2 proteins, there are clear differences between plants and cyanobacteria at the stromal ends of membrane-spanning α-helices, even though these proteins are highly homologous. The differences in subunit arrangements in these two complexes may reflect an adaptation to the peripheral antenna systems, membrane-extrinsic phycobilisomes in the case of cyanobacteria and membrane-bound peripheral LHCII subunits in the case of green plants (Büchel and Kühlbrandt 2005).
State transitions are a physiological adaptation mechanism that changes the interaction of the peripheral antenna proteins with the PSI and PSII core complexes. In green plants and green algae, membrane-bound peripheral antenna proteins play a specific mobile role to (re)direct part of the excitation energy either towards PSI or PSII ; whereas in cyanobacteria the phycobilisomes are considered to play this role. It was found with FRAP measurements that the phycobilisomes diffuse quite rapidly, but that PSII is immobile on the timescale of the measurement, indicating that the linkage between PBSs and the photosystems is unstable (Sarcina et al. 2001, 2006; Joshua et al. 2005). Based on these data it was proposed that the lateral diffusion of PBSs is involved in regulation of photosynthetic light-harvesting (state 1-state 2 transitions) (Mullineaux et al. 1997). It was found that phycobilisomes diffuse, on average, nearly three times faster in a PsaL(-) mutant than in the wildtype (Aspinwall et al. 2004). It is currently believed that light-induced state transition depends completely on a movement of PBSs towards PSI or PSII (Li et al. 2006).
Despite the fact that high-resolution data on the interaction of PBSs and photosystems are lacking, models for their organization have been presented (Bald et al. 1996). For hemidiscoidal phycobilisomes the key features are the PSII dimer as prerequisite for interaction and a row-like organization of PSII dimers as a template for phycobilisome attachment. Two PSII monomers form the ‘contact area’ within the PSII-PBS supercomplex under so-called ‘state 1’ conditions. In this model attachment of PBS with Photosystem I (PSI) under so-called ‘state 2’ conditions was proposed (Bald et al. 1996). This model stresses that the multimeric state of the PSI and PS II complexes will be of relevance. In cyanobacteria, PSI can be present as monomers or trimers and PSII as monomers or dimers. In red algae, both PSI and PSII are different because they bind several copies of membrane-associated antenna copies, which are lacking in cyanobacteria (S. Kereïche, R. Kouřil, G.T. Oostergetel, A. B. Doust, E.J. Boekema, J.P. Dekker, unpublished data). Moreover, PSI only exists as a monomer (Gardian et al. 2007).
We have studied the supramolecular conformation of hemiellipsoidal PBSs in Porphyridium cruentum, as well as—for comparison—hemidiscoidal PBS in a Synechocystis PCC 6803 mutant by single particle electron microscopy. PBS-thylakoid vesicles provide relatively uniform PBS structure in contrast to isolated PBSs, and allow us to acquire a spatial view of hemiellipsoidal structure. It is suggested that one PBS is likely to associate and line-up with one PSII dimer, in both cyanobacteria and red algea despite the fact that the red algal PBSs are two times larger in width. The larger PBS width in red algae can, however, be compensated by a membrane-bound peripheral antenna, absent in cyanobacteria.
Materials and methods
Isolation of isolated PBSs and PBS-thylakoid membranes
Porphyridium cruentum was grown in an artificial sea water medium. Flasks were supplied with 3% CO2 in air through a plug of sterile cotton at a constant temperature of 18°C. Cultures were illuminated continuously with light provided by daylight fluorescent lamps at different light intensities, 6 Wm−2 (low-light) and 15 Wm−2 (high-light). The separation of isolated PBSs followed a published protocol (Glazer 1988).
The PBS-membrane vesicles were prepared as described by Mustardy et al. (1992) with slight modifications. Cells were harvested by centrifugation (5000 g; 10 min), rinsed with 0.5 M potassium phosphate buffer (pH 7.0), and then suspended in SPC medium (0.5 M sucrose, citrate, 0.5 M potassium phosphate, 0.3 M potassium citrate, pH 7.0). After homogenization, the cells were broken in a French press at 4,000 psi in order to achieve large membrane sheets with minimal disturbance. The broken cell mixture was layered on a two-step sucrose gradient (1.0 M and 1.3 M) with the same buffer conditions as the cell suspension. After centrifugation for 30 min in a micro-centrifuge, the PBS-containing thylakoids were collected from the 1.0–1.3 M sucrose interface.
Samples for imaging were prepared by placing glow-discharged, collodion-carbon-coated copper grids (200 mesh/inch) onto a droplet of sample solution for 10 min, excess liquid was then blotted off and the samples were fixed with 2% glutaraldehyde, followed by successive washes on three droplets of double-distilled water. Staining was achieved by placing the grid on a droplet of 2% uranyl acetate. Finally, the staining solution was blotted off.
Electron microscopy
Electron microscopy was performed on a CM120 electron microscope (Philips, Eindhoven, The Netherlands) operated at 120 kV. Images were recorded under low dose conditions (a total dose ∼25 e−/Å2) with a 4,000 SP 4 K slow-scan CCD camera (Gatan, Pleasanton, CA). Images of 2-D arrays of membrane-bound phycobilisomes were recorded at 45,000 magnification, at a defocus value of −600 nm with a pixel size of 6.5 Å at the level of the specimen, (after binning the images). From the sample of non-crystalline phycobilisomes, 600 images were recorded at 80,000 magnification, at a defocus value of −380 nm with a pixel size of 3.75 Å at the level of the specimen after a binning of 2. In total, about 12,000 single particle projections were selected for single particle image analysis, which was performed with the Groningen Image Processing (GRIP) software package on a PC cluster. Selected single-particle projections were aligned by multi-reference and reference-free alignment procedures (Penczek et al. 1992; van Heel et al. 2000). Particles were then subjected to multivariate statistical analysis followed by hierarchical classification (van Heel et al. 2000).
Results
Membranes with attached PBSs were studied with electron microscopy. By cross-linking with gluteraldehyde, the integrity of the PBS organization on the membrane was preserved. Under low-light growth conditions (6 W/m−2), the PBSs on the membrane were mostly arranged in ordered domains with a high packing density of about 560 ± 20 PBSs per μm2 (Fig. 1, left frame). In contrast, at higher light intensities the distribution of PBSs is largely disordered, and the density is significantly lower, 384 ± 45 PBSs per μm2 (Fig. 1, right frame). This is in agreement with earlier reports of the dependence of pigment density in P. cruentum on growth conditions (Jahn et al. 1984; Gantt et al. 2003), but this is the first time that it is shown that the PBS arrangement is also affected.
Fig. 1Typical examples of negatively stained cyanobacterial membranes and associated phycobilisomes showing multiple ordered 2-D arrays under low-light conditions (left frame) and a random distributiom under high-light conditions (right frame) and the space bar equals 1 μm for the left frame and 400 nm for the right frame
Single particle analysis was performed on large sets of electron microscopy projections, selected from EM specimens with negatively stained membranes, on which many PBSs were still membrane-bound and smaller numbers lying around the membranes. The best preserved details were found in the top view of the membrane-bound PBS, which has maximal dimensions of 60 × 41 nm in projection (Fig. 2A). Free PBSs have about the same features but lack sharp details (Fig. 2B). A trapeziform projection shows the PBS in a side view position, but it appears rather fuzzy, despite the fact that over 4,000 projections were processed (Fig. 2C). Smaller projections are almost featureless (Fig. 2D, E). A small, almost circular-shaped projection with 3-fold symmetry and a diameter of about 11.5 nm is a common impurity and is very likely to be a PBS fragment released during storage or EM specimen preparation (Fig. 2F). Presumably this is a single disc of phycobiliproteins with a linker polypeptide incorporated as can be discerned from the electron density in the centre.
Fig. 2Single particle electron microscopy of phycobilisome projections from Porphyridium cruentum (A) View in the membrane plane of membrane-bound phycobilisomes (B) View in the membrane plane of free phycobilisomes (C–E) side views of free phycobilisomes. (F) Most common phycobilisome fragment. (G) image of A with 2-fold rotational symmetry imposed after analysis (H) Image of C, with mirror symmetry imposed after analysis. The number of summed projections for A-F is 1520, 484, 4096, 380, 499 and 746, respectively. The space bar is 25 nm
In an attempt to study the types of packing of PBSs within 2D arrays we compared several hundred areas. In parts with a low amount of ordering the PBSs have a tendency to associate into specific linear structures with their long edges. This is also true for those arrays with the highest amount of ordering. These membrane parts do not show a uniform type of packing, as is exemplified by two average sums of arrays showing a different repeating motif and size, which is about 55 × 35 nm in Fig. 3C and 45 × 31 nm in Fig. 3D, respectively.
Fig. 3Comparison of semi-crystalline arrays of phycobilisomes. (A) the membrane-attached phycobilisome (B) APC core structure of Synechocystis PCC 6803 ck mutant, sum of 5019 projections for comparison (A.A. Arteni, R. Ajlani, E.J. Boekema, unpublished results). (C) 2D array with semi-crystalline phycobilisomes, an average of 11 fragments (D) 2D array with semi-crystalline phycobilisomes, an average of 107 fragments (E) PSII dimer (from Thermosynechococcus elongatus) (F) PSII double dimer, found after solubilization of membranes with dodecyl maltoside, a sum of 59 projections (G) PSII double dimer, found after solubilization of membranes with digitonin, a sum of 46 projections. The space bar is 50 nm
Since there may be specific interactions of the PBSs and the underlying membrane arrangement of PSII, we also compared these arrays with the dimensions of PSII and with those of a hemi-discoidal type PBSs of cyanobacteria. The width of cyanobacterial PBSs can be derived accurately from the central PBS component, which is the APC core structure. In the top view position, the APC core structure has a length and width of 22.5 and 15 nm, respectively (Fig. 3B). The width is about the same of a PSII core dimer (Fig. 3E). During previous structural studies on PSII from Thermosynechococcus elongatus we have observed small numbers of a higher type of association of PSII, which were double dimers (Fig. 3F). These dimers are also present in low numbers after membrane solubilization with digitonin, without further purification steps (Fig. 3G; Folea and Boekema, unpublished data).
Discussion
Single particle electron microscopy revealed the overall features of the hemispherical PBS of Porphyridium cruentum. The PBSs attached to the membranes have the best preserved features, whereas the side views of free PBSs appear fuzzy, indicating some amount of structural flexibility. Some of the flexibility could have been caused by the air drying of the negatively stained EM specimens. On the other hand, the packing of two types of 2D arrays also differs, because the total surface in one type of packing is substantially smaller than in the other one (Fig. 3), suggesting intrinsic flexibility.
The supramolecular organization of the red algal and cyanobacterial membranes is not understood in detail, because we do not know how the PBSs are precisely associated to PSII and PSI. There is for instance no information on the subunits involved in this interaction. Some low-resolution interaction concepts are based on freeze-fracture electron microscopy, which has revealed that cyanobacterial PSII can be arranged in a row-like organization in the membrane with a repeat of 10–12 nm (Mörchel and Schatz 1987) or 14 nm (Nilsson et al. 1992). The double dimers presented in Fig. 3 and first discussed in Kuhl et al. 1999, are considered to be remnants of the row-like PSII organization. Based on these data, a model for the arrangement of PBSs was proposed as described in Bald et al. (1996). In this model the PBSs associate along the rows with their short axis as a repeat. This model makes sense for photosynthetic organisms with hemi-discoidal PBSs because there is a 1:1 match between the 15 nm width of the APC core, as presented in Fig. 2, and the PSII dimmer, which has about the same width.
The situation in the red alga P. cruentum is, however, obviously different because of the much larger size of its PBSs. It could have a different or no special arrangement of its PBSs in relation to row-like PSII. In particular it was shown that the tricylindrical core of the PBSs in P. cruentum is two times wider than that of the hemidiscoidal forms in cyanobacteria (Redecker et al. 1993), which we confirm in the current study. This is commensurate with the suggestion that the PBSs in red algae bind to a tetrameric PSII complex, on the basis of freeze-fracture EM imaging (Redecker et al. 1993; Teckos et al. 2004). Further corroboration is derived from the stoichiometry of PBSs and PSII complexes which is about 1:3–4 (Cunningham et al. 1989). These authors show that the stoichiometry of the photosystems and PBSs in P. cruentum is largely unaffected by light intensity (Cunningham et al. 1989).
However, we have to take into account that red algal PSI and PSII have a peripheral membrane-embedded antenna system that makes the total PSI and PSII supercomplexes substantially larger in surface area. The precise arrangement of the peripheral antanna in red algal PSII is unknown, but it was recently found that in the related unicellular cryptophyte Rhodomonas SC 24 the peripheral antenna extends the width of PSII by 50% on one of its sides (S. Kereiche, R. Kouřil, G.T. Oostergetel, A. B. Doust, E.J. Boekema, J.P. Dekker, unpublished data, see Appendix I). If such an antenna arrangent would also exist on both sides of P. cruentum PSII, the width would have been doubled to about 30 nm, and become similar to the PBS width in one of the 2-D arrays (Fig. 3D). This would mean that a 1:1 ratio between rows of PSII and PBSs is also possible in red algea, despite the giant dimensions of the PBSs. This would be a meaningful ratio for light-harvesting under low-light conditions, where ordered arrays have been observed. But whether a 1:1 or a lower 1:2–4 ratio is true or not needs to be established by further work on the supramolecular organization of PSII within the membrane and by analyzing the size and shape of supercomplexes composed of PSII and its peripheral antenna by single particle electron microscopy.
Electronic supplementary material
Below is the link to the electronic supplementary material.
(DOC 156 kb)

phycobilisome
red alga
porphyridium cruentum
electron microscopy
single particle analysis


The Role and Limitations of 18-Fluoro-2-deoxy-d-glucose Positron Emission Tomography (FDG-PET) Scan and Computerized Tomography (CT) in Restaging Patients with Hepatic Colorectal Metastases Following Neoadjuvant Chemotherapy: Comparison with Operative and Pathological Findings

Background Recent data confirmed the importance of 18-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) in the selection of patients with colorectal hepatic metastases for surgery. Neoadjuvant chemotherapy before hepatic resection in selected cases may improve outcome. The influence of chemotherapy on the sensitivity of FDG-PET and CT in detecting liver metastases is not known.
Introduction
The liver is the most common, and often the only, site of distant metastases from colorectal cancer (CRC).1 Hepatic resection is the only effective therapy for a subset of patients with CRC metastatic to the liver, and is associated with 5-year survival rates ranging from 25 to 40%.2–5 From 60 to 65% of patients will, however, develop recurrent tumors after hepatic resection, indicating that they had harbored unrecognized intra- or extrahepatic tumor foci at the time of liver resection.6 Moreover, several studies report unresectable disease in 40–70% of patients that undergo laparotomy for liver resection.2,7,8 These data indicate that better patient selection is needed to avoid unnecessary operations. There are several potential ways of improving patient selection, one of which is the administration of neoadjuvant therapy followed by reevaluation and better preoperative staging.
Positron emission tomography with the glucose analog 18-fluoro-2-deoxy-d-glucose (FDG-PET) is a sensitive diagnostic tool for the detection of colorectal metastases. Approximately 25% of patients are discovered to have new intra- or extrahepatic tumors on FDG-PET performed after standard imaging.9–13 Screening with FDG-PET before hepatic resection for CRC significantly improves the survival rates of resected patients, probably by improving patient selection.14
The role of neoadjuvant chemotherapy to down-stage nonresectable liver metastases and to improve outcome following hepatic resection of resectable liver metastases is an evolving concept, but one that is not yet established. With the recent application of new chemotherapeutic agents, such as irinotecan, oxaliplatin, and bevacizumab, improved response rates can be achieved and the use of these agents in the neoadjuvant setting would appear to be especially relevant for patients with nonresectable disease or patients with high risk of recurrence.15–18
The aim of our study was to examine the effect of neoadjuvant chemotherapy for hepatic colorectal metastases on CT and FDG-PET/CT findings and to define the role of these imaging techniques in this setting. To do so, we compared CT and FDG-PET/CT findings with histopathological reports.
Patients and Methods
Patients
Patients with colorectal liver metastases were assigned to receive either an immediate liver resection (group 1) or neoadjuvant chemotherapy (group 2). The criteria for neoadjuvant treatment were:
Nonresectable tumors due to size, location, and number and assessment of the surgical team that complete (R0) resection was not technically possible.High risk of recurrence according to the Memorial Sloan–Kettering Cancer Center (MSKCC) clinical risk score to assess risk of recurrence.19 Specifically, patients with two or more risk factors [number of metastases >1, disease-free survival <12 months, carcinoembryonic antigen (CEA) levels >200 ng/ml, metastases from the colonic tumor to regional lymph nodes, size of the largest metastases >5 cm] were assigned to neoadjuvant treatment.Presence of extrahepatic disease.Oncologist’s preference—this applied to patients with MSKCC >2 that were referred from other hospitals for immediate surgery. The decision not to administer neoadjuvant therapy was not necessarily in agreement with our policy.Patient’s preference—patients who refused neoadjuvant therapy were assigned to immediate surgery when feasible.
Neoadjuvant Chemotherapy
Treatment consisted of a neoadjuvant chemotherapeutic combination of 5-fluorouracil, leucovorin, and either oxaliplatin (FOLFOX) or irinotecan (FOLFIRI). Seventeen patients (35%) were also given bevacizumab. Most of the group 2 patients were given neoadjuvant irinotecan unless they were enrolled on a multicenter study whose protocol consisted of the administration of neoadjuvant oxaliplatin.
Staging
Before undergoing neoadjuvant chemotherapy, all group 2 patients underwent a triphasic contrast-enhanced CT scan, and a FDG-PET/CT was performed in 30 (62.5%) of them. All 75 patients in group 1 and group 2 underwent FDG-PET/CT and abdominal CT before liver surgery. The time interval between the last course of chemotherapy and the FDG-PET/CT scan was at least 2 weeks, and surgical exploration took place within 1 month following the FDG-PET/CT scan in most of the cases. Because we used an integrated PET/CT technique, precise anatomical localization could be achieved and confirmed with the standard triphasic abdominal CT findings.
PET/CT
The patients were asked to fast for at least 4 h before undergoing PET/CT. Earlier lab tests had shown that they all had glucose levels <150 mg%. The patients received an intravenous injection of 370–666 MBq (10–18 mCi) of 18F-FDG. Data acquisitions by an integrated PET/CT system (Discovery LS; GE Medical Systems, Milwaukee, WI, USA) were performed within 60–120 min after injection. Iodinated oral contrast material was given to opacify loops of the bowel on the CT image. Data acquisition was as follows: CT scanning was performed first, from the head to the pelvic floor, with 140 kV, 80 mA, a tube rotation time of 0.5 s, a pitch of 6, and a 5-mm section thickness, which was matched to the PET section thickness. Immediately after CT scanning, a PET emission scan that covered the identical transverse field of view was obtained. Acquisition time was 5 min per table position. PET image data sets were reconstructed iteratively by applying the CT data for attenuation correction, and coregistered images were displayed on a workstation (Xeleris, Elgems, Haifa, Israel).
Studies of all patients were retrieved and read in consensus by two experts (U.M. and E.E.-S.). All suspected sites of metastatic disease showing an increased FDG uptake were recorded. The location of hepatic lesions was recorded according to the Couinaud segmental classification.
Hepatectomy
All patients without extensive extrahepatic disease underwent surgical exploration and intraoperative ultrasound (IOUS). Resections of all metastatic sites were performed by either anatomic or R0 nonanatomic resection, with a tendency toward maximal parenchymal preservation with nonanatomic resections.
Complete radiological response to neoadjuvant chemotherapy was defined as the complete resolution of all metastatic sites according to the CT and PET-CT. In these cases, careful palpation and IOUS were performed in search of remaining tumor or scarring. When there was no evidence of either, the tumor sites were resected according to the findings on the original imaging (i.e., before any response to neoadjuvant treatment).
Detection of Hepatic Metastases
To define the sensitivity of CT and FDG-PET/CT for liver metastases, imaging results were compared with the presence and size of liver lesions as demonstrated and measured by histopathological reports.
Results
Patients
Between June 2002 and September 2005, 75 patients with 155 suspected metastatic lesions from a primary CRC underwent hepatic resection in our department. Group 1 included 27 patients with 33 lesions who underwent immediate liver resection and group 2 included 48 patients with 122 lesions who first received neoadjuvant chemotherapy before subsequently undergoing liver resection. The patient’s profiles are outlined in Table 1. Table 2 lists the operative procedures that were performed in the two groups.
Table 1Study Patients’ Profiles Group 1 (n = 27)Group 2 (n = 48)P valueSex ratio (F/M)0.500.920.22Mean age, years (std deviation)66 (9.8)61.25 (10.9)0.06Site Colon9 (71%)32 (66%)0.74 Rectum8 (29%)16 (33%)LN metastases (Duke’s >B in colonic specimen)81.5%82%0.73No. of liver tumors (mean) (std deviation)1.19 (0.4)2.52 (1.9)0.0001Max tumor diameter (largest) (std deviation)3.53 cm (2.84)3.9 cm (1.84)0.49Extrahepatic disease (no. of patients)790.56Prior liver resection461Mean MSKCC risk score (range)1.82 (0–4)2.48 (2–5)0.003Group 1, immediate hepatic resection; Group 2, hepatic resection following neoadjuvant chemotherapyLN = lymph nodeTable 2Operative ProceduresOperative procedure (no. of patients; lesions)Group 1(n = 27)Group 2 (n = 48)Right hepatic lobectomy58Left hepatic lobectomy44Central hepatectomy03Right trisegmentectomy10Nonanatomic resections1529Left lat segmentectomy22Explorative laparotomy (no resection)02Group 1, immediate hepatic resection; Group 2, hepatic resection following neoadjuvant chemotherapy
Detection of Hepatic Metastases
The overall findings, the sensitivity, specificity, and accuracy of triphasic contrast-enhanced CT and FDG-PET/CT in the detection of viable liver metastases compared to the pathological results are presented in Table 3. FDG-PET and CT had a statistically significant higher sensitivity in detecting liver metastases in patients who did not receive chemotherapy compared to patients who received chemotherapy (Table 3). Statistical analysis also revealed that triphasic contrast-enhanced CT had a higher sensitivity than PET/CT in detecting colorectal metastasis following neoadjuvant treatment (65.3 vs 49%, respectively, P < 0.0001), but not in patients who did not receive neoadjuvant therapy (87.5 vs 93.3%, P = 0.625).
Table 3FDG-PET and CT—Comparison With Pathological Results Group 1 n = 33Group 2 n = 122P valuePET TP2948 True negative (complete response)–20 FP24 FN250 Sensitivity93.3%49%<0.0001 Specificity–83.3%CT TP2864 True negative (complete response)–18 FP16 FN434 Sensitivity87.5%65.3%0.038 Specificity–75%Group 1, immediate hepatic resection; group 2, hepatic resection following neoadjuvant chemotherapy
Four of the six false-positive (FP) results on FDG-PET involved patients who had previously undergone hepatic resection. These lesions were discovered on follow-up FDG-PET/CT. Uptake was observed along the resection site, and these patients underwent nonanatomic liver resections for suspected locally recurrent lesions. Pathologic evaluation failed to reveal any tumor cells. The positive predictive value of FDG-PET/CT for metastasis recurrence in the resection site was only 33%, and specificity was 60%.
Sensitivity of FDG-PET in the detection of colorectal metastasis correlated with the size of the metastasis (Table 4). Average size of the metastases in the two groups was 33.9 mm (standard deviation 19) in group 1 and 18.9 mm (standard deviation 19) in group 2, P < 0.0001.
Table 4Sensitivity of FDG-PET: Correlation With Tumor SizeTumor size<1 cm1–3 cm>3 cmGroup 1 sensitivity (total no. of lesions)33% (n = 3)100% (n = 15)92% (n = 13)Group 2 sensitivity (total no. of lesions)17% (n = 35)78% (n = 41)100% (n = 22)Group 1, immediate hepatic resection; group 2, hepatic resection following neoadjuvant chemotherapy
We also compared the sensitivity of CT and FDG-PET for patients who received FOLFIRI or FOLFOX (n = 31) with patients who received the same regimen plus bevacizumab (n = 17). The results are outlined in Table 5. We found that the sensitivity of FDG-PET, but not of CT, was lower in patients who received bevacizumab, although the difference did not reach statistical significance.
Table 5FDG-PET and CT in Patients who Received Chemotherapy With or Without Bevacizumab: Comparison With Pathological Results Bevacizumab −Bevacizumab +P valuePET TP2919 True negative (complete response)173 FP22 FN2030 Sensitivity59%39%0.068CT TP3331 True negative (complete response)135 FP60 FN1618 Sensitivity67%63%0.9
Detection of Extrahepatic Metastases
In group 1, there were one FP result for extrahepatic disease (suspected recurrence in colonic anastomosis, abdominal wall), one true-positive (TP) result (recurrence in mesocolic lymph nodes), and one false negative (FN) result (in a patient with peritoneal metastases). In group 2, there was one FP result (for suspected peritoneal metastasis), three TP results (recurrence in paraaortic lymph nodes and solitary lung metastasis), and two FN results (for peritoneal metastases).
Discussion
The role of neoadjuvant chemotherapy followed by hepatectomy for colorectal liver metastases has not yet been clearly established. New chemotherapeutic agents, including irinotecan, oxaliplatin, and the biologic agent bevacizumab, have yielded improved response rates in the treatment of advanced CRC. These agents may have a potential role in the neoadjuvant setting for down-staging both nonresectable disease to resectability15,16 and resectable disease, probably mostly for patients with high risk of recurrence.17 Our policy is to administer neoadjuvant treatment to patients with nonresectable disease, those with extrahepatic disease, and those with resectable disease who have two or more risk factors according to the MSKCC clinical risk score.19 One of the theoretical benefits of neoadjuvant treatment is that patients who develop additional extrahepatic or intrahepatic metastases during this time period are spared a futile major operative procedure. Accurate staging before the beginning of neoadjuvant treatment and restaging following the treatment are crucial for optimal patient selection.
The standard preoperative staging of patients with colorectal liver metastases includes combined abdominal CT and chest x-ray or chest CT. It was recently demonstrated that FDG-PET as a complementary staging method improves the therapeutic management of patients with colorectal liver metastases.20 Preoperative screening with FDG-PET results in an increased survival rate of patients who undergo liver resection.14 This can be explained by the detection of occult intra- and extrahepatic metastatic disease, thus obviating futile explorations. In the current study, the sensitivity of FDG-PET/CT following neoadjuvant therapy was only 49% compared to a sensitivity of 93.3% in patients who did not receive neoadjuvant treatment (P < 0.0001). The influence of the chemotherapeutic drugs on the sensitivity of FDG-PET in detecting extrahepatic metastases is not known, but we could assume that it is influenced in a similar way. This may result in a higher-than-expected rate of nonresectable disease discovered at the time of laparotomy and more extrahepatic recurrences following resection. In our series, only three of the 48 patients (6.25%) who received neoadjuvant chemotherapy were found to have nonresectable disease (one had diffuse liver metastases and two had peritoneal spread) that was not discovered preoperatively by either abdominal CT or FDG-PET/CT. We believe that one of the reasons for the high operability rate is the fact that a significant number of patients underwent a baseline FDG-PET/CT before the administration of neoadjuvant chemotherapy. We therefore recommend performing a baseline FDG-PET scan for all candidates for liver resection before the administration of neoadjuvant treatment. A longer follow-up is needed to assess the results of our application of this protocol.
The decreased sensitivity of FDG-PET/CT in detecting liver metastases should also be a consideration when planning the extent of liver resection. We believe that the extent of resection should be guided by additional imaging modalities, including abdominal CT and IOUS, in patients who received neoadjuvant treatment. In our series, triphasic contrast-enhanced abdominal CT had a higher sensitivity than FDG-PET/CT in detecting colorectal metastasis in patients who received neoadjuvant chemotherapy (65.3 vs 49%, P < 0.0001). The higher sensitivity of CT alone compared to FDG-PET/CT in detecting small colorectal metastasis has been reported by Ruers et al.,20 and this may be even greater in patients who received chemotherapy. An attractive solution is the integrated PET/CT scanner on which a diagnostic triphasic abdominal CT scan can be performed at the same setting as the PET scan.
There are several possible explanations for the decreased sensitivity of FDG-PET/CT in the detection of colorectal metastases following neoadjuvant therapy:
Size of the lesion. The sensitivity of FDG-PET in detecting colorectal metastasis was reported as being directly related to the size of the lesions.20 We found similar results in our series (Table 4). The average size of the metastases following neoadjuvant treatment was significantly smaller than that in patients who did not receive chemotherapy (33.9 mm in group 1 and 18.9 mm in group 2, P < 0.0001). Two FN results in group 1 and 32 in group 2 involved tumors smaller than 1 cm. We can assume that one of the main reasons for the decreased sensitivity of FDG-PET following chemotherapy is the decrease in size of the metastases.Chemotherapy and “metabolic shutdown.” It has been demonstrated that the sensitivity of FDG-PET is diminished in cancer patients who undergo the examination less than 2 weeks following the administration of chemotherapy,21 presumably due to a temporary metabolic “shutdown.” Although the scans in our study were done with a minimal interval of 2 weeks from the last course of chemotherapy, partial response to therapy may have caused decreased FDG uptake in metastatic lesions, making them undetectable in comparison to the physiological background uptake of FDG in the liver. This may have been a contributing factor to the FN results in our series. We found a lower sensitivity of FDG-PET (but not of CT) in detecting liver metastases following regimens including bevacizumab compared to regimens that did not include bevacizumab, although the difference did not reach statistical significance. This result may have significant clinical implications; however, it needs to be verified in larger series.Time interval between the FDG-PET and surgery. In our series, two patients with FN results (two hepatic lesions) underwent surgery more than 2 months after FDG-PET was performed. Viable tumors were discovered at the site of the original metastases which had disappeared on FDG-PET following neoadjuvant treatment. Although it is conceivable that the relatively long interval between FDG-PET and surgery may have contributed to the FN results, we believe that these tumors may have been FN due the small size of the lesions following partial pathological response to chemotherapy.Nonavid tumors. PET avidity of the tumors can be assessed only in patients who undergo a baseline FDG-PET before neoadjuvant treatment. It has been reported that FDG-PET is less sensitive for mucinous adenocarcinoma.22 In our series, ten FN results (lesions) were in patients with nonavid mucinous adenocarcinoma (sensitivity 37.5%).
There were two FP results in group 1 and four FP results in group 2. Four of the six FP results were in patients who had undergone a previous hepatic resection, for which follow-up FDG-PET detected uptake in the same location of the resected metastasis. These patients underwent nonanatomic resections of the “lesions.” The pathological examination revealed only foreign body reaction without any tumor cells. In our current study, FDG uptake in the tumor bed following previous resection had a positive predictive value of 33% (2/6). The specificity of FDG uptake in the tumor bed for recurrence was 60%. We believe that FDG uptake in the tumor bed following a previous liver resection is not specific for tumor recurrence, especially if the CEA levels are normal. Nguyen et al. demonstrated that FDG uptake may be high in various granulomatous lesions,23 possibly explaining the FP results along resection margins. Therefore, biopsy or follow-up should be considered in these cases.
Conclusions
The sensitivity of FDG-PET in detecting colorectal hepatic metastases decreases significantly following neoadjuvant chemotherapy. This may result in a higher-than-expected rate of nonresectable disease discovered at the time of laparotomy and in more extrahepatic recurrences following resection. We recommend staging patients with a “baseline” contrast-enhanced FDG-PET/CT both before and after the administration of neoadjuvant therapy. The extent of hepatic resection should be guided by systematic integration of data from all additional imaging modalities (abdominal CT, IOUS), as well as by the original imaging findings (before the neoadjuvant treatment). We recommend resection of all metastases that achieved complete radiological response, whenever technically possible. Longer follow-up and further studies are required to justify neoadjuvant treatment and screening with FDG-PET/CT in patients with colorectal metastases to the liver who are at high risk of recurrence.

fdg-pet
neoadjuvant chemotherapy
colorectal liver metastases


Variations in 123I-metaiodobenzylguanidine (MIBG) late heart mediastinal ratios in chronic heart failure: a need for standardisation and validation

Background There is lack of validation and standardisation of acquisition parameters for myocardial 123I-metaiodobenzylguanidine (MIBG). This lack of standardisation hampers large scale implementation of 123I-MIBG parameters in the evaluation of patients with chronic heart failure (CHF).
Introduction
Radiolabelled metaiodobenzylguanidine (123I-MIBG), an analog of the false neurotransmitter guanethidine, localises in adrenergic nerve terminals primarily via the norepinephrine (NE) transporter (uptake-1) system [1, 2]. In the past two decades, a large number of investigators have demonstrated decreased myocardial 123I-MIBG uptake in heart failure patients and have shown that those with the lowest uptake tend to have the poorest prognosis [3–11]. There have also been findings suggesting that abnormalities of myocardial 123I-MIBG uptake may be predictive of increased risk for ventricular arrhythmia and sudden cardiac death (SCD) [12, 13].
One factor that has constrained acceptance of cardiac 123I-MIBG imaging as a clinical patient management tool in heart failure has been the variability of technical aspects of the procedure. Although most publications have included the heart-to-mediastinum ratio (H/M) as the measure of myocardial uptake, the methods used to obtain this parameter show considerable variation. In addition, the influence of procedural and acquisition parameters on the reproducibility of this measurement technique have only occasionally been considered. While the effect of collimator selection on H/M ratios has been studied, similar attention has not been paid to the impact of administered activity, acquisition time and duration [14]. Given the absence of published guidelines for cardiac 123I-MIBG imaging, standardisation of procedures among individual users of this agent becomes even more important.
The objective of the present study was to examine the influence of procedural variables associated with 123I-MIBG imaging when a standardised technique was used to determine the H/M ratio.
Materials and methods
Study design
Myocardial 123I-MIBG scintigrams on 290 heart failure patients (male and female subjects ≥18 years of age with New York Heart Association [NYHA] Classes II–IV) from six sites in five European countries were retrieved and analysed. These imaging studies were performed between 1993 and 2002 and were collected for a previously described trial [15]. This trial was designed to show that standardised methodology could be applied to the analysis and processing of 123I-MIBG myocardial images to obtain reproducible quantitative results. As the large majority of patients had only late planar 123I-MIBG images, the analysis of these images is the sole focus of this paper.
Investigational sites sent copies of all digital image files to an Imaging Core Laboratory (ICL) for evaluation and analysis. Data on amount of 123I-MIBG activity injected and image acquisition parameters (i.e. delay after injection of 123I-MIBG and start of acquisition, acquisition duration and type of collimator used) were collected.
The primary efficacy objective of the original trial was to determine the prognostic significance of the late H/M ratio in relation to occurrence of major cardiac events (MCE), defined as either: 1) cardiac death due to all causes, including myocardial infarction (MI), progressive heart failure and sudden cardiac death (SCD); 2) cardiac transplant; 3) potentially fatal arrhythmic event, including resuscitated cardiac arrest or appropriate internal cardiac defibrillator (ICD) discharge. Results of that analysis have been published. For the purposes of this paper, H/M thresholds associated with low-, intermediate- and high-risk for MCEs were defined to provide the basis for the multivariate analysis described below.
Image analysis
An experienced nuclear medicine technologist not affiliated with the ICL processed all the planar images to determine the heart/mediastinum (H/M) ratio. The heart region of interest (ROI) was drawn manually to include both ventricles and any atrial activity that was clearly visible. A square mediastinal ROI was drawn in the upper mediastium, using the apices of the lungs as anatomic landmarks (Fig. 1). The H/M ratio was calculated as the ratio of the counts/pixel in the two ROIs.
Fig. 1Example of processing procedure for late planar 123I-MIBG images. The positioning of the mediastinal ROI was standardized in relation to the lung apex, the lower boundary of the upper mediastinum and the midline between the lungs
Statistical analysis
Data are presented as mean ± standard deviation, unless indicated otherwise. Kaplan–Meier survival analysis and the log-rank test were used to analyse differences in event rate for different subject groups defined by the late H/M thresholds. Differences between these groups for continuous data were compared using analysis of variance (ANOVA) with a post hoc Bonferroni. Categorical data were compared using the Fisher’s exact test. A forward stepwise multivariate regression analysis was performed to determine independent predictors of late H/M. Explanatory variables were related to patient characteristics (i.e. gender, age, NYHA class, left ventricular ejection fraction [LVEF], and underlying etiology of heart failure) and to acquisition parameters (i.e. amount of injected 123I-MIBG activity, collimator type, delay after injection and start of acquisition, and duration of acquisition). Multivariate forward stepwise regression analysis was performed for all study subjects. The overall goodness-of-fit for each model was expressed as the adjusted R2. The F test was used to assess whether a model explained a significant proportion of the variability. A p < 0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed with SPSS (SPSS for Windows, version 11.01, SPSS Inc, Chicago, IL, USA).
Results
As previously described, there were 67 major cardiac events. Figure 2 shows a Kaplan–Meier survival analysis for three different groups of late H/M. Quantitative estimates for low risk (late H/M >2.0, 2-year survival of 95%), intermediate risk (late H/M 1.4–2.0, 2-year survival of 76%) and high risk (late H/M <1.4, 2-year survival of 45%) could be determined. The differences in event rate were statistically significant between the three groups (all p < 0.001).
Fig. 2Kaplan–Meier curve showing the relation between survival and time to first major cardiac event for three groups with different H/M values: late H/M <1.4 vs late H/M 1.4–2.0, p < 0.0001; late H/M<1.4 vs late H/M >2.0, p < 0.0001; late H/M 1.4–2.0 vs late H/M>2.0, p < 0.0001
Table 1 shows the baseline characteristics and the parameters related to the 123I-MIBG acquisition of all subjects and for the three different groups of late H/M. Overall, the majority of subjects were male and were in functional class II according the NYHA classification and had a LVEF<50% (n = 262). Between the three late H/M groups, there were differences in age (subjects with a late H/M >2.0 were younger), NYHA classification, LVEF (increasing from subjects with a late H/M <1.4 to subjects with a late H/M >2.0) and HF etiology. In addition, there were no differences in parameters of heart failure (LVEF and NYHA classification) between the different centres.
Table 1Patient and scintigraphic parameters for all subjects and subdivided by late H/M 123I-MIBG All (N = 290)Late H/M<1.4 (N = 51)1.4–2.0 (N = 143)>2.0 (N = 96)Patient characteristics Male/Female237/5339/12*117/26*81/15* Age (years)53 ± 11 (21–83)55 ± 10‡54 ± 10‡49 ± 11 NYHA (II/III/IV)165/116/911/34/6†85/55/3†69/27/0† LVEF (%)32 ± 14 (7–85)23 ± 9†31 ± 13‡39 ± 14* Etiology of heart failure (isch/non-isch)121/16920/31‡70/73‡31/65‡Scintigraphic parameters Collimator type (LEGP/LEHR/ME)50/216/24†24/25/2†24/104/15†2/87/7† Activity (MBq)164 ± 53 (72–370)145 ± 31†173 ± 64160 ± 41 Delay after injection (h)4.3 ± 0.6 (1.9–6.9)4.1 ± 0.5‡4.3 ± 0.5‡4.5 ± 0.6 Duration of acquisition (min)9.6 ± 3.3 (1–15)11.3 ± 4.3†9.2 ± 3.49.4 ± 2.0*Data are presented as mean ± standard deviation (range).NYHA New York Heart Association, LVEF left ventricular ejection fraction, Isch ischemic, Non-isch non-ischemic, LEGP low-energy all purpose, LEHR low-energy high resolution, ME medium energy, MBq mega-Becquerel, h hours, min minutes.Categorical data (Fisher’s exact test, for each group): *p = 0.53; †p < 0.001; ‡p < 0.035.Continuous data (ANOVA with posthoc Bonferoni): *p < 0.01 compared to late H/M <1.4; †p < 0.01 compared to late H/M 1.4–2.0; ‡p < 0.01 compared to late H/M >2.0.
Overall, most acquisitions were performed with low-energy high-resolution (LEHR) collimators (74%), followed by low-energy general purpose (LEGP) collimators (17%). Only 9% of acquisitions were made with medium-energy (ME) collimators, all from one centre. The amount of activity injected ranged from 72 to 370 MBq (mean 164 ± 53 MBq). There was a large variation in the time-span after between injection of 123I-MIBG and imaging, ranging from 1.9 up to 6.9 h (mean delay 4.3 ± 0.6 h). The duration of acquisition could be classified in seven categories: 1, 2, 3, 4, 5, 10 and 15 min. The majority of acquisitions was performed with a 10-min duration (n = 203). There were differences between the three late H/M groups in collimator type, amount of injected 123I-MIBG, delay after injection, and duration of acquisition (Table 1).
Table 2 shows the results of the stepwise multivariate regression analysis for all subjects and all parameters. LVEF, collimator type, duration of acquisition, NYHA classification and age were independent predictors of late H/M. This model predicted approximately 30% of the variation in late H/M results (p < 0.0001). Even after exclusion of the very low acquisition durations (<5 min), the duration of acquisition remained an independent predictor of late H/M.
Table 2Multivariate analysis of clinical and acquisition related parameters as predictors of late H/M for all subjects (N = 290)VariablesCoefficient bStandard error bP value Constant0.5490.411 LVEF0.0150.02<0.001 Collimator type0.5310.087<0.001 Duration of acquisition0.0450.0140.001 NYHA−0.1210.0550.028 Age−0.0060.0030.034Goodness-of-fit of the model: adjusted R2 = 0.312; P value = <0.0001
In subjects with a late H/M <1.4, stepwise multivariate regression analysis showed that NYHA classification and delay after injection were the only independent predictors of late H/M (Table 3). This model predicted approximately 10% of late H/M (p = 0.03). In subjects with a late H/M 1.4–2.0, stepwise multivariate regression analysis showed that LVEF and collimator type were the only independent predictors of late H/M (Table 4). This model predicted approximately 10% of late H/M (p = 0.001). In subjects with a late H/M >2.0, duration of acquisition was the only independent predictor of late H/M (Table 5). However, duration of acquisition predicted only 1% of late H/M and explained a statistically non-significant proportion of variability (p = 0.347).
Table 3Multivariate analysis of clinical and acquisition related parameters as predictors of late H/M for subjects with a late H/M<1.4 (N = 51)VariablesCoefficient bStandard error bP value Constant1.6800.161 NYHA−0.0550.0270.049 Delay after injection−0.0680.0300.031Goodness-of-fit of the model: adjusted R2 = 0.104; P value = 0.030Table 4Multivariate analysis of clinical and acquisition related parameters as predictors of late H/M for subjects with a late H/M 1.4–2.0 (N = 143)VariablesCoefficient bStandard error bP value Constant1.3700.078 LVEF0.0040.0010.001 Collimator type0.0860.0300.005Goodness-of-fit of the model: adjusted R2 = 0.100; P value = 0.001Table 5Multivariate analysis of clinical and acquisition related parameters as predictors of late H/M for subjects with a late H/M >2.0 (N = 96)VariablesCoefficient bStandard error bP value Constant0.0221.376 Duration of acquisition0.1190.0570.040Goodness-of-fit of the model: adjusted R2 = 0.013; P value = 0.347
Discussion
In a heterogeneous population of heart failure patients, the most important determinant of cardiac uptake of 123I-MIBG is the degree of impairment of the NE transporter function in presynaptic adrenergic neurons [3–11]. As such, considerable variation in late H/M would be expected between patients with otherwise similar clinical presentations (NYHA class, LVEF, etc). Nevertheless, the findings of the present study indicate that a proportion of the variation in the late H/M 123I-MIBG can also be explained by differences in acquisition parameters. Of particular interest, the factors contributing to this variation differed for different ranges of late H/M.
Abnormalities in myocardial 123I-MIBG imaging have been demonstrated in patients with both common cardiac pathologies such as CAD, CHF, and non-ischemic cardiomyopathy [3–12, 16–18] and more uncommon entities such as Syndrome X [19], Brugada Syndrome [20] and idiopathic ventricular fibrillation [13]. However, there has been limited standardisation of the acquisition and quantitative analysis techniques used by various investigators, and most studies have involved small numbers of subjects and locally defined reference standards and measures of outcome. Thus, despite the large number of studies on cardiac 123I-MIBG imaging, these methodological and analytical limitations have hampered the implementation of this technique for the evaluation and management of individual patients.
Acquisition duration was one of the independent predictors of late H/M. The positive correlation suggests that a longer acquisition time is associated with a higher late H/M. However, it is more likely that this association reflects that a longer acquisition time results in a higher number of counts and the allied improvement of count statistics. In particular, in patients with reduced myocardial uptake (i.e. heart failure patients), the higher signal-to-noise ratio for a longer acquisition (≥10 min in the present study) plays a pivotal role in the accurate assessment of late H/M. In addition, delay after injection was in high-risk subjects (late H/M <1.4), an independent predictor of late H/M. The negative correlation suggests that a longer acquisition time is associated with a lower late H/M.
However, in analogy to acquisition duration, it is more likely that this association reflects that a longer delay results in a lower number of counts and the allied deterioration of count statistics. In particular, in patients with reduced myocardial uptake (i.e. heart failure patients), the higher signal-to-noise ratio for a shorter delay after injection plays a pivotal role in the accurate assessment of late H/M.
The most well-validated influence on the measured late H/M is the collimator type. Because of the high-energy photons emitted by 123I, septal penetration affects estimation of the H/M ratio in 123I-MIBG imaging with a low-energy (LE) collimator [21]. Medium-energy (ME) collimators have been shown to improve quantitative accuracy in 123I studies [14, 21, 22]. While these results would suggest that semi-quantitative cardiac 123I-MIBG imaging might best be performed using ME collimators, there are practical limitations to such a recommendation. Almost all nuclear cardiology procedures are now performed on a multi-head gamma camera, and many dedicated dual-head cardiac cameras are not supplied with ME collimators (at least for SPECT).
The precise impact of collimator choice on the prognostic value has not been evaluated yet. However, it seems feasible that the improved accuracy of the ME collimators will decrease the number of patients with an intermediate risk likelihood and thereby improving the prognostic accuracy of myocardial uptake parameters of 123I-MIBG. It is therefore important to establish the degree to which the collimator used affects prognostic utility of the H/M measurement.
Rigorous and uniform analysis of cardiac 123I-MIBG parameters minimises inter-and intra-individual variation [15, 23]. In the analysis of the entire population, numerous variables (LVEF, collimator type, duration of acquisition, NYHA classification and age) contributed to the prediction of H/M, but only two of these variables are under the control of the imager. Furthermore, the present results suggest that the severity of cardiac sympathetic neuronal dysfunction also modulates the impact of acquisition parameters on the H/M determinations. For example, in the low-risk group (H/M >2.0), none of the parameters had a meaningful impact on the numerical result, indicating that preserved myocardial uptake of 123I-MIBG can be reliably determined under widely different acquisition conditions. By comparison, considering user-controlled variables, assessment of high-risk subjects (H/M <1.4) was affected by delay after injection, while the assessment of H/M in the intermediate risk subjects was affected by the collimator employed. Standardisation therefore is most important in assessing patients with more severe disease, although in the present study the procedural variables only contributed a relatively small fraction of the overall variability in H/M determinations.
Aging is associated with changes in the cardiovascular autonomic regulation, both under resting and stimulated conditions. At rest, elderly subjects show increased sympathetic and decreased parasympathetic activity as well as impaired arterial baroreflex function [24–34]. The negative correlation, although weak, between age and late H/M is in line with the finding that sympathetic activity increases with age typically reflected by increased circulating catecholamine levels, and that it is associated with decreased myocardial uptake of 123I-MIBG, an observation previously reported by Somsen et al. [35] and Estorch et al [36].
123I-MIBG has been shown by individual centres to have both good reproducibility and acceptable variability, and to have prognostic value in CHF patients [3–11, 23, 37, 38]. These single-centre experiences, however, do not necessarily allow for extrapolation of the obtained results to other centres. In spite of this, quantitative thresholds are often implemented without inter-institutional validation. Because there are differences between centres in hardware, acquisition parameters and post-acquisition processing, the extrapolation to other centres, or generalisation of these single-centre findings, is not per se justified. Especially due to the lack of well-defined data sets, validation in each separate centre and between centres has not been possible.
Recently, a realistic 3D-gated cardiac phantom, the Amsterdam gated cardiac phantom (AGATE phantom), was developed and validated [39]. Such a phantom could be used as a tool for the validation in each separate centre and for standardisation between centres. However, a phantom can only partially simulate patient characteristics. Therefore, the phantom results obtained will give only an impression of repeatability and variability, but they do not necessarily reflect the accuracy of measurements in patients. Therefore, in addition to the phantom measurements, a set of reference patient studies should also be included in a validation of the technique [40]. Moreover, the results should be validated in each separate centre and monitored for reproducibility and consistency.
Several medications used to treat patients with heart failure may influence myocardial 123I-MIBG uptake. However, in our study the data on medication use were relatively complete, available for about 80% of the study subjects, so any further analysis could only be based on this subset. The problem with pursuing a medication analysis is that any effect on 123I-MIBG uptake is related to individual physiology and efficacy of treatment rather than on the technical attributes of the imaging, which was the main focus of this manuscript. In addition, the main manuscript already included results examining these effects, specifically that subjects that had events were less likely to be on β-blockers than those who did not have events [15]. A specific analysis of the effect of medication was not deemed appropriate to this manuscript. In addition, the use of medication that interferes with myocardial uptake of 123I-MIBG (such as tricyclic antidepressants), was not significant and did not warrant a separate analysis.
The primary limitation of the present study has already been noted, namely, that the largest influence on the H/M is cardiac disease status rather than image acquisition parameters. Although collimation and acquisition duration were independent predictors of late H/M, their individual contributions to measurement variance were <10% each. Because of the retrospective nature of this multi-centre study, it was not possible to control for the effects of numerous variables that might have had undetected impact on late H/M. Studies using phantoms have already established the effect of collimator selection on H/M, and similar studies could be performed to examine the influence of count rate and total counts. Additional patient studies that included multiple acquisitions of different durations and times post-injection could establish the effect of these parameters on H/M with greater clarity.
In conclusion, the limited standardisation in acquisition parameters reported in literature was also reflected in the results of this multi-centre study. A wide variety of collimators, delay after injection of 123I-MIBG to the start of the acquisition and acquisition durations were reported. In general, validation and standardisation require commitment and perseverance. Moreover, without proper validation and standardisation, the role myocardial 123I-MIBG in the clinical management of CHF will continue to be constrained. Without these requirements, the use of myocardial 123I-MIBG will be limited to a single centre research tool for understanding the physiology of myocardial sympathetic neuronal activity under different conditions and after interventions. With application of appropriate standardisation, myocardial 123I-MIBG imaging could become a routine procedure for evaluation of heart failure and other cardiac disease populations.

mibg
heart failure
prognosis
instrumentation


Insulinoma misdiagnosed as juvenile myoclonic epilepsy

We report a girl presenting with drug-refractory myoclonia and generalized tonic-clonic seizure (GTCS) on awakening who, initially, was misdiagnosed as having juvenile myoclonus epilepsy (JME) before insulinoma was detected.
Many conditions may mimic or cause epileptic seizures, including psychiatric and movement disorders, cardiac arrhythmias and metabolic abnormalities. Toxic and metabolic causes are potentially curable, are refractory to antiepileptic drugs, and may be fatal if untreated. Insulinoma is a rare and treatable cause of hypoglycemic seizures which might be misdiagnosed as intractable epilepsy.
Case report
In May 2001 a 13-year-old girl was referred to the hospital because of GTCS on awakening. The family history was unremarkable and her personal history revealed one simple febrile seizure at age 3 years. One month before admission two episodes of confusional states were reported which lasted 5 and 30 min and were characterized by unresponsiveness and psychomotor slowing.
Physical and neurological examinations were normal, weight was 62 kg (97th centile), height 162 cm (90th centile), and menarche was at age 13 years (March 2001, 1 month before the first seizure). Awake EEG the day after GTCS revealed normal background activity, bilateral synchronous and asynchronous high-amplitude delta waves over the parietotemporal regions, and right frontal sharp-slow waves. Blood count, blood chemistry (glucose 4.5 mmol/l, calcium 2.4 mmol/l, potassium 4.0 mmol/l), and magnetic resonance imaging (MRI) of the brain were normal. Confusional states were interpreted as complex partial seizures and GTCS as a secondary generalization. Low-dose carbamazepine (6.3 mg/kg per day) was introduced. Two weeks later, a second GTCS on awakening occurred, followed by two short episodes of unresponsiveness before breakfast in front of the television. Another few days later, perioral and eyelid myoclonus and jerks in the arms and legs were noticed early in the morning. After sleep deprivation a series of myoclonic jerks at awakening in the morning and after several naps were registered coinciding with generalized low-amplitude spikes or polyspikes. JME was diagnosed and carbamazepine replaced by valproate (20 mg/kg per day) which was not effective. Blood glucose was normal (4.2 mmol/l 4 h after food intake). Two months later (August 2001) a second overnight long-term video-EEG revealed generalized discharges of spikes or polyspikes and slow waves in the second part of the night. Again myoclonic jerks in the hands and legs with a single generalized low-amplitude spike and slow waves were registered on awakening (Fig. 1).
Fig. 1Ictal EEG on awakening: generalized low-amplitude spike with simultaneous registration of myoclonic jerks of the hands and legs. Although the EEG pattern is not so typical for JME, together with the history and the clinical symptoms this EEG seemed compatible with this diagnosis
In September 2001, 6 months after the manifestation of the first seizures, low preprandial blood glucose (1.8 mmol/l, normal 3.8–6.1) and high insulin (31 μU/ml, normal 1.9–2.3) were detected because the patient was sluggish and dizzy (glucose and insulin were measured by routine methods). Inappropriate insulin secretion was suspected and confirmed by pre- and postprandial glucose and insulin measurements (preprandial: glucose 2.8 mmol/l, insulin 33 μU/ml and 50 min after food intake: glucose 3.1 mmol/l, insulin 153 μU/ml); therefore, a 72-h fasting test was not done. Abdominal ultrasound (US) and MRI revealed a round and circumscribed hypoechoic lesion (2.1 × 1.8 × 1.9 cm) with sharp but uneven borders in the pancreatic body without evidence of metastases. Insulinoma was suspected. Because multiple endocrine neoplasia type I syndrome (MEN I) is associated in 5–10% of the cases with insulinoma [5, 9], prolactin, thyroid-stimulating hormone, parathyroid hormone, and thyroxin (T3, T4) were determined, which were all normal; therefore, mutation analysis of exons 2–10 of the MENIN gene was not done. Valproate was stopped and the patient was advised to have food intake every 2 h and three times during the night. Since then she has been seizure free, but myoclonic jerks recurred occasionally when meals were delayed by >5 h during the night. Her weight had increased from 62 to 84 kg within the last 10 months before surgery.
Surgery was performed 5 months later (March 2002) in Moscow and two insulinomas were revealed by intraoperative US: one located on the superior-posterior surface between the head and body (diameter 2.5 cm) and the other on the anterior surface of the pancreas (diameter 0.7–0.9 cm). Before the surgery, US and MRI disclosed only one lesion. Both insulinomas were removed in toto and the endocrine pancreas was suppressed by Sandostatin (octreotide) for 4 days. Histology confirmed the diagnosis of two benign beta-cell adenomas. The patient fully recovered, remained seizure free during the next 4 years, has a current weight of 62 kg (75th centile), and is mentally normal.
Discussion
Myoclonus and GTCS after awakening are hallmarks of JME, especially when adolescents are affected [2]. Hence, in our patient the interpretation of the clinical symptoms together with spike waves was indicative of JME. Confusional states and drug-refractory seizures on the other side are less compatible with the diagnosis of JME, but fit well with hypoglycemic events. The latter, however, were erroneously thought to be excluded by two normal blood glucose measurements. Furthermore, hypoglycemia in nondiabetic adolescents is much rarer than JME. Insulinoma, responsible for the profound hypoglycemia in our patient, has its onset usually in middle [10] or older age [4] and is very rare in children [9]. Symptoms are prominent following prolonged fasting states, especially in the morning as in our patient, but also in the late afternoon [4]. Hypoglycemia can cause loss of consciousness, sluggishness, confusion, asthenia, deep coma, dizziness, disturbances in vision, and epilepsy. An international review of 1,067 cases [10] showed such neuropsychiatric symptoms in 92% of the patients. Irreversible damage of the central nervous system occurred in 6.8% of the cases [10]. Forty-one percent of the patients have amnesia during the hypoglycemic event [4]. Therefore, the history has to be obtained from family members or friends. Clinical details about the onset, duration, and type of symptoms as well as temporal association with fasting and the relief of symptoms after food intake are helpful information for making the diagnosis. Considerable weight gain as in our patient was reported in 39% of the patients [4]. Taking into account that an actually normal glucose level does not exclude episodes of hypoglycemia, a prolonged fasting test up to 72 h under close monitoring is recommended to assess inappropriate insulin secretion [3].
The median interval from the onset of symptoms to the diagnoses of insulinoma is 2 years, with a wide range of 1 month to 30 years [4]. Initial misdiagnosis is frequent. In a series of 59 patients with insulinoma, prior diagnosis included neurological disorders in 39 (64%) and especially epilepsy in 23 (39%) patients. Seven patients (12%) were treated with antiepileptic drugs [4]. The seizure symptoms are variable. During severe hypoglycemia generalized and focal seizures occurred in children [6]. In patients with insulinoma, shaking of all four limbs at night [8], jerking and twitching [1], and symptoms suspicious for partial and complex partial seizures with and without secondarily generalized seizures are reported [3, 5]. Varying degrees of EEG changes and sometimes massive spiking were recorded during hypoglycemia [7]. In patients with insulinoma, focal and widespread EEG slowing [1, 3] combined with epileptic activity [5] are mentioned.
Conclusions
The combination of seizures and episodic confusional states should prompt the search for hyperinsulinism, especially when the episodes occur in fasting states and when they are refractory to antiepileptic treatment. Inappropriately high insulin with low blood glucose is diagnostic; this constellation has to be investigated resolutely (e.g., by the classic 72-h fasting test) but with caution under strict clinical and chemical monitoring. Timely diagnosis of an insulinoma is of paramount importance to prevent sequelae for such patients.

insulinoma
juvenile myoclonic epilepsy
myoclonus
confusional state
hypoglycemia


The effect of stimulation technique on sympathetic skin responses in healthy subjects

The aim of this study was to collect normative data for sympathetic skin responses (SSR) elicited by electrical stimulus of the ipsilateral and contralateral peripheral nerves, and by magnetic stimulus of cervical cord. SSRs were measured at the mid-palm of both hands following electrical stimulation of the left median nerve at the wrist and magnetic stimulation at the neck in 40 healthy adult volunteers (mean age 52.2 ± 12.2 years, 19 males). The onset latency, peak latency, amplitude and area were estimated in “P” type responses (i.e., waveforms with a larger positive, compared to negative, component). SSR onset and peak latency were prolonged when the electrical stimulus was applied at the contralateral side (i.e., the SSR recorded in the right palm P < 0.001). The onset latency was similar on both sides during cervical magnetic stimulation. However, peak latency was faster on the left side (P < 0.03). Comparison of electrical and magnetic stimulation revealed that both the onset and peak latency were shorter with magnetic stimulation (P < 0.001). The latency of a SSR varies depending on what type of stimulation is used and where the stimulus is applied. Electrically generated SSRs have a longer delay and the delay is prolonged at the contralateral side. These factors should be taken into account when interpreting SSR data.
Introduction
Sympathetic skin response (SSR) testing is commonly used to assess sympathetic function [18]. Abnormal SSR occur in both central [7, 8] and peripheral autonomic dysfunction [1, 16, 21].
The SSR is a complex polysynaptic reflex, which courses through a common centrifugal path via sympathetic post-ganglionic unmyelinated fibers [2, 24]. However, its exact pathway through the central nervous system is not fully understood. A variety of stimuli can generate a SSR, which illustrates the complexity of the afferent inputs to the reflex.
Although measuring SSR is relatively simple, interpreting these measurements is not so straightforward. Different afferent stimuli may well lead to a wide range of normal SSR values. It is difficult to attribute abnormalities of SSR to sympathetic dysfunction alone, particularly in cases of polyneuropathy, due to the potential influences by somatic fibers sensory inputs [24].
The aim of this study was to collect normative SSR data. SSRs were measured in both mid-palms following electrical stimulation of the left medial nerve and magnetic stimulation of the neck in healthy controls. SSR onset and peak latency, amplitude and area were analyzed. Electrically generated SSR in the ipsilateral (left) and contralateral (right) palms were compared to the SSR evoked by magnetic stimulation.
Methods
Subjects
Forty healthy adult volunteers [9 males/21 females, mean age 52.21 ± 12.23 (range 28–75) years, height 172 ± 11.3 cm] were studied. All subjects were healthy, with no history of cardiovascular or neurological disease. All subjects denied previous alcohol or drug abuse. None of the subjects had contraindications for magnetic stimulation, such as metal prosthesis or pacemaker. All participants gave written informed consent prior to the start of the study.
Procedure
The study was performed using a 2-channel Counterpoint, Medtronic-Dantec (Medtronic-Dantec Electronics, Skovlunde, Denmark) electromyographic unit. SSR recordings were based on the technique described by Shahani et alal. [20]. Briefly, subjects lay supine in an air-conditioned, quiet room with temperature maintained at 24°C and skin temperature at 33 ± 0.5°C. The same investigator performed all experiments, and care was taken to avoid all external stimulation. Active standard disk ECG Ag/AG/Cl electrodes were attached to the mid-palm of both hands and referenced to the corresponding dorsal surfaces. Skin temperature was verified as being above 32°C by measuring close to the recording electrodes. The recording sites were the same in all experiments.
Electrical stimulation
Single square-wave electrical pulses of 0.1 milliseconds duration were applied to the left median nerve at the wrist at irregular intervals (30–60 seconds) to produce a SSR. The sweep speed was 1,000 milliseconds/D, the amplifiers sensitivity was 100 μV/div and the amplifier filters were set at 0.5 Hz and 2 kHz for the low and high frequency filter, respectively. Five trials were recorded simultaneously from both palms and on each side the response with the largest amplitude and shortest latency were taken. A fixed stimulus intensity of 60 mA was used throughout.
Magnetic stimulation
A Magstim 200 stimulator, equipped with a circular coil of 9 cm inner diameter and a 2.0-T maximum magnetic field strength, was used to apply magnetic stimulation at the cervical area. The center of the circular coil was placed in the midline over the 7th cervical spinous process. The current was set to anti-clockwise flow in the coil (side A visible) and the stimulus intensity fixed at 60% of maximum output. Five stimuli were delivered randomly every 20–30 seconds.
Analysis of waveforms
To avoid habituation 5 minutes rest was given between electrical and magnetic stimulation. To maintain uniformity of the results only “P” type responses (waveforms with a larger positive, compared to negative, component) were analyzed.
Figure 1 shows how the components of the waveform were analyzed. The onset latency was taken from the time of the stimulus artefact to the start of the positive deflection. The peak latency was taken from the time of the stimulus artefact to the peak of the positive deflection. The peak-to-peak amplitude and the area under the SSR curve were also measured.
Figure 1A typical sympathetic skin response recording. Typical SSR recordings taken from the right palm (upper trace) and left palm (lower trace) (a) following electrical stimulus of left median nerve and (b) magnetic stimulation of cervical cord
Statistical analysis
Differences between stimulus procedures in the same subjects were determined by using paired t-tests. The tests were two tailed. Statistical significance was set at P < 0.05. All analyses were performed using SPSS for Windows (10.0; SPSS Inc., Chicago, IL).
Results
Clear SSR recordings with a main positive component (i.e., P waveforms) were obtained in all participants.
Latency
Mean SSR onset and peak latency on the right side, which was contralateral to the electrical stimulus, was significantly prolonged compared to the left side, which was ipsilateral to the electrical stimuli (P < 0.001, Tables 1 and 2). No differences in the onset latency were found between right and left sides following cervical magnetic stimulation. However, a weak interside difference in peak latency (P < 0.03) was observed (faster on the left side). Comparison between electrical and magnetic stimulus revealed that both onset and peak latency on either side were significantly shorter for magnetic stimulation (P < 0.001, Tables 1 and 2).
Table 1Normative sympathetic skin response data in healthy subjectsElectrical ipsilateral (L)Electrical contralateral (R)Magnetic (L)Magnetic (R)Latency-onset (seconds)1.79 ± 0.221.90 ± 0.24a1.63 ± 0.25c1.66 ± 0.28dLatency-peak (seconds)2.54 ± 0.332.68 ± 0.38a2.40 ± 0.27c2.43 ± 0.28b,dAmplitude (mV)2.67 ± 1.492.35 ± 1.32a2.74 ± 1.612.70 ± 1.69Area (mV seconds)2.85 ± 1.532.63 ± 1.413.24 ± 1.993.22 ± 1.96dAll data are mean ± SD. Electrical stimulation was applied at the left medial nerve of the wrist. SSR were recorded in both the left (ipsilateral) and right (contralateral) palms. Statistically significant difference (P < 0.05)a Electrical stimulus—between sidesb Magnetic stimulus—between sidesc Left side—between electrical and magnetic stimulusd Right side—between electrical and magnetic stimulusTable 2The difference in sympathetic skin response onset latencies evoked by electrical and magnetic stimulationLatency difference (milliseconds)Electrical stimulation. R–L105 ± 85 (0–320)*Magnetic stimulation. R–L27 ± 86 (−110–330)Left electrical–magnetic158 ± 183 (−50–640)*Right electrical–magnetic237 ± 201 (0–780)*Differences in SSR onset latency measurements during 3 types of stimulation (left and right electrical stimulation and magnetic stimulation). All data are mean ± SD (and range). L, left; R, right. * Significance of P < 0.05 (comparison by one sample t-test)
Amplitude and area
During electrical stimulation amplitude was higher of the stimulated (left) side compared to the non-stimulated side. Amplitude was similar during magnetic and contralateral electrical stimulation. The area of the SSR measured at the right side was greater for magnetic stimulation compared to electrical stimulation.
Discussion
The main finding of this study was that the latency of a SSR depends on whether an electrical or magnetic evoking stimulus is used. We also found a mean latency difference between contralateral and ipsilateral evoked electrical responses of 105 milliseconds.
SSR are generated through a complex somato-sympathetic reflex with spinal, bulbar and suprabulbar components [13]. Until now, they have not been precisely defined in humans [26]. SSR can be evoked by various types of stimuli including auditory, deep breathing, painful sensation of the limbs or face etc., and are therefore mediated by different afferent pathways [2]. Moreover, SSR are evoked by impulses that originate in a different area to which the response is produced. For example, electrical pulses over the sternal skin can be used to elicit SSR in the palms [19], which highlight the complexity of the internal neuronal network involving polysynaptic relays and connections between different vertical levels along the neuraxis and in both sides of the human body [11]. It is, therefore, not surprising that measurement of a SSR depends on the site and the type of stimulus used to initiate the reflex.
The longer latency in the contralateral side of 105 milliseconds may be explained by the crossing over of the signal from one side of the body to the other. The cross over time of around 100 milliseconds suggests that the reflex circuits are supraspinal [13]. Data from animal models indicate that the crossing occurs at multiple levels, including the brainstem and spinal cord [26]. However, patients with cervical or high thoracic lesions resulting in complete tetraplegia or paraplegia have no SSR from the hands and feet [6], suggesting that supraspinal centers are essential in the generation of SSR.
A constant latency to alternatively electrical stimulation of median nerves was suggested in a previous study [3]. However, latency was measured with 20 consecutive electrical stimulations, and thus the effects of adaptation (or habituation) cannot be excluded.
Magnetic stimulation may be most reproducible method to evoke SSR [14]. In theory, strong stimulation generated by the magnetic field close to the spine avoids any possible interference by affected somatic sensory fibers, providing a measure of efferent sympathetic function alone. In the case of the lower motor neurons, it has been showed that cervical magnetic stimulation excites the motor roots at or very near the exit foramen or even further distally [5]. In respect of SSRs, magnetic stimulation could, in analogy to motor neurons, excite the sensory roots at their entry [14] or directly activate the sympathetic trunks within the spinal canal [25].
In our study, the latency of the SSR evoked by magnetic stimulation was shorter than the corresponding values of the electrically elicited responses. The shorter latency of the magnetically generated SSR could be caused by impulses bypassing the peripheral afferent part of the reflex. Given that the difference between electrical stimulation at the wrist and magnetic stimulation at the neck was 158 milliseconds and assuming that the average distance from wrist to C7 is 70 cm, the conduction velocity of afferent somatic fibers is calculated to be approximately 5 m/second.
Previous studies suggested that the efferent path is served by sympathetic B and C fibers conducting at approximately 2 m/second [12, 17], whereas the afferent path is served by somatic myelinated sensory groups II and III conducting at rates of 50–5 m/second [12]. In good agreement with our results, other investigators have reported a latency difference between electrically and magnetically elicited SSR of 80 milliseconds [14] and 130 milliseconds [4], with the former being always longer.
Based on our results, it can be assumed that the SSR onset latency to spinal cord magnetic stimulation was the shortest, whereas that to contralateral peripheral nerve electrical stimulation was the longest. Furthermore, the difference in latency between SSR to magnetic and ipsilateral electrical stimuli was close to that between SSR to ipsilateral and contralateral electrical stimuli. A satisfactory explanation for the observed differences would not be offered without the risk of being highly speculative.
The morphology is another element of SSRs instability. Two main types of SSR waveform were previously recognized; P type which has a larger positive component and N type with a larger negative component [15, 22, 23]. It has been shown that strength of stimulation was positively correlated with P type amplitude and inconsistently with N type and that strong stimuli probably produced P type responses [22]. To ensure similarity of measurements and facilitate comparison of responses to different kinds of stimuli, we arbitrarily choose to study only subjects with predominant P type responses. In none of our participants the change of stimulation from electrical (ipsilateral and contralateral) to magnetic resulted in switching of P to N type of SSR. This can be the consequence of using relatively high intensity in electrical and magnetic stimulus.
The latency values in our subjects were slightly higher than those in previous reports. In those reports, the mean SSR latency from the palm to electrical stimulation of the median nerve ranged between 1.27 seconds and 1.51 seconds [4, 10, 14, 24]. This discrepancy should be attributed to a methodological difference. The negative onset of the response is generated by the electrical activation of the sweat gland. The peak of the negative phase is probably variable and generated by the relationship between sweat production and the surround tissue [24]. Under the particular recording conditions applied, the majority of the examined individuals had potentials with an initial positive peak. Therefore, instead of measuring the onset latency to the first negative peak, which is most often used, we measured the onset latency from stimulus artifact to the onset of the main positive wave irrespective of the existence or not of a proceeding small negative component. As regards to the peak latency values, they showed similar to onset latency differences in this study. Thus, peak latency is not suggested as an additional parameter, which could be useful in clinical practice.
Amplitude is the SSR parameter that has been reported to be highly variable. It can be influenced by several factors such as temperature, time of the day, emotion and for this reason several authors have not used it as measure of disease [2, 9]. The relative stability of amplitude and area measurements among the SSRs evoked by different stimuli in our subjects is noticeable. These findings could be attributed to the stable conditions of recordings (single session for all SSR recordings in each subject and long interstimulus interval to avoid habituation).
There are certain potential limitations in the study design. First, the fact that SSRs were not assessed for reproducibility in individual subjects. However, the phenomenon of habituation [3] did not allow us to compare measurements (particularly for the amplitude) of consecutive responses in a single individual.
Second, since it is known that a variety of stimuli are capable of producing SSR, startle could intermix with the electrical stimulus and, mostly, auditory could interfere with the magnetic stimulus, leading to shorter SSR latencies. A study comparing parameters of SSR elicited by pure auditory as opposed to other types of stimulation should clarify this point. However, in a previous study, where ear-plugs were used to exclude auditory stimuli, the SSR latency measurements to neck magnetic stimulation were even shorter than in our subjects [14], suggesting that the intermixed auditory stimulation is not a major limitation. Finally, habituation, in a broad sense including the central processing and familiarity, cannot be completely ruled out by a 5-minute interval between techniques, although intervals of 40–60 seconds between trials were reported elsewhere [10, 14, 22]. Application of electrical followed by magnetic stimulation in some subjects and the inverse procedure in others might have been used, although this would have increased the complexity of the study design.
In brief, SSRs’ have been introduced as an easy to perform and painless mean for the evaluation of post-ganglionic sympathetic function [20]. In the filed of autonomic nervous system where the diagnostic options are limited, and despite the expression of certain doubts, mainly related to the large diversion of individual values, SSR remains at present the most widely applied neurophysiological test for the sympathetic nervous system. According to the results of the present study, latency which is the most widely quoted parameter, varied depending on the stimulation type, being shorter at ipsilateral than contralateral electrical stimulus and even less short when magnetic stimulation at the neck was applied. Further studies on the challenging issue of SSR waveform and its possible clinical relevance are necessary to explore it in detail.

sympathetic skin response
methods
autonomic nervous system
galvanic skin response
sudomotor function