R.W.F. de Bruin

Monitoring In Situ Dosimetry and Protoporphyrin IX Fluorescence Photobleaching in the Normal Rat Esophagus During 5-Aminolevulinic Acid Photodynamic Therapy¶

Experimental therapies for Barretts esophagus, such as 5‐aminolevulinic acid (ALA)–based photodynamic therapy (PDT), aim to ablate the premalignant Barretts epithelium. However, the reproducibility of the effects should be improved to optimize treatment. Accurate irradiation with light of a proper wavelength (633 nm), fluence and fluence rate has shown to be critical for successful ALA‐PDT. Here, we have used in situ light dosimetry to adjust the fluence rate measured within the esophagus for individual animals and monitored protoporphyrin IX (PpIX) fluorescence photobleaching simultaneously. Rats were administered 200 mg kg−1 ALA (n = 14) or served as control (n = 7). Animals were irradiated with an in situ measured fluence rate of 75 mW cm−2 and a fluence of 54 J cm−2. However, this more accurate method of light dosimetry did not decrease the variation in tissue response. Large differences were also observed in the dynamics of PpIX fluorescence photobleaching in animals that received the same measured illumination parameters. We found that higher PpIX fluorescence photobleaching rates corresponded with more epithelial damage, whereas lower rates corresponded with no response. A two‐phased decay in PpIX fluorescence could be identified in the response group, with a rapid initial phase followed by a slower rate of photobleaching. Nonresponders did not show the rapid initial decay and had a significantly lower rate of photobleaching during the second phase of the decay (P= 0.012).

Systematic review of sarcopenia in patients operated on for gastrointestinal and hepatopancreatobiliary malignancies

Preoperative risk assessment in cancer surgery is of importance to improve treatment and outcome. The aim of this study was to assess the impact of CT‐assessed sarcopenia on short‐ and long‐term outcomes in patients undergoing surgical resection of gastrointestinal and hepatopancreatobiliary malignancies.

Use of fibre optic probes for detection of Barrett’s epithelium in the rat oesophagus by Raman spectroscopy

In the last decades, there has been a dramatic increase in the incidence of Barretts oesophagus and the associated oesophageal adenocarcinoma. Therefore, patients with a Barretts oesophagus undergo regular endoscopic surveillance with randomly taken biopsies to detect the presence of high-grade dysplasia or carcinoma. Sampling errors and observer variation, inherent to such a surveillance protocol warrant other detection methods. Raman spectroscopy is a non-invasive optical spectroscopic technique that provides detailed information about the molecular composition and structure of tissues. Changes in molecular composition in tissues as a consequence of pathologic processes, can thus be recognised. For clinical application of Raman spectroscopy, thin and flexible fibre optic probes can be used that fit in the auxiliary channel of an endoscope. In this study, a multivariate classification model was developed for detection of Barretts epithelium, based on ex vivo Raman spectra of the rat oesophagus. The spectra were collected with three different fibre optic probes and on 11 different days. This mimics the way a database is collected in a clinical situation, including all instrument calibration, probe-to-probe and day-to-day variation. After elimination of interfering background signal contributions from the different probes using a vector-correction procedure, we could discriminate between Barretts and normal epithelium with accuracy higher than 93%. The model yields a spectral discriminant that best separates the two groups. To facilitate interpretation of this discriminant, we obtained Raman spectra from tissue sections of normal oesophageal epithelium, keratin and muscle layer using a confocal Raman microscope.

Anastomotic leakage, the search for a reliable biomarker. A review of the literature.

Background  Colorectal anastomotic leakage (AL) is a severe complication leading to severe infection, sepsis and sometimes death. At present the diagnosis is made clinically, usually at 6–8 days after surgery. An objective biomarker reflecting the intra‐abdominal milieu surrounding the anastomosis would be a useful additional diagnostic tool to make the diagnosis of AL before its clinical presentation. This review aims to assess the current status of the search for such a biomarker in peritoneal fluid.

Modulation of tumor growth by allogeneic blood transfusion

SummaryThe effect of a single blood transfusion on the formation and outgrowth of experimental lung metastases was assessed in two tumor models in rats. The transfusions were given either 1 week before (day-7) or 1 week after (day +7) tumor cell inoculation. The first approach was performed to investigate the effect of transfusions on the formation of lung colonies, the second approach to study the effect on the outgrowth of established metastases. The first tumor model used was a transplantable, nonimmunogenic sarcoma (LS175) in BN rats. Animals were injected i.v. with 105 tumor cells and the number of metastases developing in the lungs was counted after 18 days. Experimental animals received 1 ml of allogeneic WAG blood, controls were given 1 ml of syngeneic BN blood. A single allogeneic transfusion given on day-7 had no effect on the formation of LS175 lung colonies but, when given day +7, stimulated the outgrowth of established metastases. The second tumor model was a highly immunogenic transplantable basal cell carcinoma (BC 1618) in inbred WAG rats. Rats were injected i.v. with 106 tumor cells and the numbers of lung colonies were counted after 21 days. Experimental animals were transfused with 1 ml of BN blood, controls received 1 ml of WAG blood. An allogeneic transfusion on day-7 led to a significant inhibition of lung metastases, whereas a transfusion on day +7 had no effect. The results clearly indicate that allogeneic blood transfusions can modulate tumor growth and metastasis. Although immunological factors seem to play a crucial role in this transfusion phenomenon, there was no clear-cut correlation between the observed effects (accelerated tumor growth vs inhibition of metastasis) and the type of immunomodulation evoked.

The Importance of Portal Venous Blood Flow in Ischemic‐Type Biliary Lesions after Liver Transplantation

Ischemic‐type biliary lesions (ITBL) are the most frequent cause of nonanastomotic biliary strictures after liver transplantation. This complication develops in up to 25% of patients, with a 50% retransplantation rate in affected patients. Traditionally, ischemia‐reperfusion injury to the biliary system is considered to be the major risk factor for ITBL. Several other risk factors for ITBL have been identified, including the use of liver grafts donated after cardiac death, prolonged cold and warm ischemic times and use of University of Wisconsin preservation solution. In recent years however, impaired microcirculation of the peribiliary plexus (PBP) has been implicated as a possible risk factor. It is widely accepted that the PBP is exclusively provided by blood from the hepatic artery, and therefore, the role of the portal venous blood supply has not been considered as a possible cause for the development of ITBL. In this short report, we present three patients with segmental portal vein thrombosis and subsequent development of ITBL in the affected segments in the presence of normal arterial blood flow. This suggests that portal blood flow may have an important contribution to the biliary microcirculation and that a compromised portal venous blood supply can predispose to the development of ITBL.

Fractionated Illumination for Oesophageal ALA-PDT: Effect on Blood Flow and PpIX Formation

Abstract. The effect of fractionating the 633 nm illumination of 5-aminolaevulinic (ALA)-based photodynamic therapy (PDT) of the normal rat oesophagus was studied. Fractionation of the illumination could enhance the PDT effect in two ways: (a) delay of the vascular shutdown or relaxation of the vasoconstriction induced by ALA-PDT and (b) use of newly formed protoporphyrin IX (PpIX), produced during the dark interval. Forty rats were randomly allocated to two groups of 20 animals each. To study vascular effects, in group 1 illumination with 633 nm (100 mW/cm) was performed at 3 h after oral ALA administration (200 mg/kg) either continuously with 20 J/cm diffuser length (n=5) or fractionated 2×10 J/cm with a 150 s interval (n=5), five animals served as controls. Blood flow was measured with a laser Doppler flowmeter. To study the effect of renewed PpIX forming, animals in group 2 were illuminated continuously at 3 h after ALA with 20 J/cm (n=5) or 40 J/cm (n=5) or fractionated 2×20 J/cm with a 3 h interval (n=5), five animals served as controls. In all animals the in vivo fluence rate and PpIX fluorescence were measured during illuminations and animals were killed at 48 h after PDT.ALA-PDT did not cause any significant vasoconstriction. Fluorescence measurements and dosimetric results in group 1 did not differ between animals illuminated continuously or fractionated with a 150 s interval. In group 2, during a 3 h dark interval, PpIX fluorescence increased and was bleached during the second illumination. The tissue optical properties changed during the 3 h dark interval, resulting in a lower in vivo fluence rate (p≤0.001). Fractionation did not result in more oesophageal damage. It was concluded that a 150 s interval during illumination in ALA-PDT does not increase oesophageal blood flow. During an interval of 3 h new PpIX is formed. In the present study, fractionated illumination using short or long time intervals did not result in more damage. Thus, this study shows no evidence for improved PDT effect with fractionated light delivery.

Improving the outcome of kidney transplantation by ameliorating renal ischemia reperfusion injury: lost in translation?

Kidney transplantation is the treatment of choice in patients with end stage renal disease. During kidney transplantation ischemia reperfusion injury (IRI) occurs, which is a risk factor for acute kidney injury, delayed graft function and acute and chronic rejection. Kidneys from living donors show a superior short- and long-term graft survival compared with deceased donors. However, the shortage of donor kidneys has resulted in expansion of the donor pool by using not only living- and brain death donors but also kidneys from donation after circulatory death and from extended criteria donors. These grafts are associated with an increased sensitivity to IRI and decreased graft outcome due to prolonged ischemia and donor comorbidity. Therefore, preventing or ameliorating IRI may improve graft survival. Animal experiments focus on understanding the mechanism behind IRI and try to find methods to minimize IRI either before, during or after ischemia. This review evaluates the different experimental strategies that have been investigated to prevent or ameliorate renal IRI. In addition, we review the current state of translation to the clinical setting. Experimental research has contributed to the development of strategies to prevent or ameliorate IRI, but promising results in animal studies have not yet been successfully translated to clinical use.

Timing of illumination is essential for effective and safe photodynamic therapy: a study in the normal rat oesophagus

Summary5-Aminolaevulinic acid (ALA)-induced, protoporphyrin IX (PpIX)-mediated photodynamic therapy (PDT) is an experimental treatment modality for (pre)malignant oesophageal lesions. This study aimed to optimize the time of illumination after ALA administration. Six groups of eight rats received 200 mg kg–1ALA orally, eight rats served as controls. Illumination was performed at 1, 2, 3, 4, 6 or 12 h after ALA administration with a 1-cm cylindrical diffuser placed in a balloon catheter (laser parameters: 633 nm, 25 J radiant energy, power output 100 mW). During illumination, fluorescence measurements and light dosimetry were performed. Animals were sacrificed at 48 h (n= 4) or 28 days (n= 4) after PDT. At day 28, an oesophagogram was performed. Largest PpIX fluorescence was found at 3 h after ALA administration. In vivo fluence rate was three times higher than the calculated incident fluence rate. At 48 h after PDT, major epithelial damage was found in all animals illuminated at 2 h, whereas less epithelial damage was found at 3–6 h and none at 1 and 12 h. In animals illuminated at 4, 6 and 12 h, but not at 2 h, oesophagograms showed severe dilatations and histology showed loss of Schwann cells. These results demonstrate that the choice of time interval between ALA administration and illumination is critical for achieving epithelial damage without oesophageal functional impairment. A short interval of 2–3 h seems to be most appropriate.

Identification of gene clusters differentially expressed during the cellular injury responses (CIR) to cisplatin

The goal of this study was to identify changes in mRNA levels in tumour cells after a toxic exposure to cisplatin (IC99dose). Using suppression-subtractive hybridization (SSH) 2 cDNA libraries were created, an UP library (202 cDNA fragments) and a DOWN library (153 cDNA fragments). Using reversed Northern hybridization 16 and 30 fragments were truly differentially expressed in the UP and DOWN libraries, respectively. Most prominent in the UP library were the mitochondrial and injury response clusters and in the DOWN library the cytoskeletal, protein synthesis and signalling clusters. These distinct clusters potentially represent an expression profile of the cisplatin-induced cellular injury response.