Introduction 6 12 22 4 9 9 4 13 16 18 19 4 4 In the present report, we provide a detailed description of the alternative trans-diaphragm approach together with a comparison with the original trans-thoracic approach. Furthermore, we illustrate its usefulness when studying the pulmonary circulation, by presenting novel comprehensive pulmonary and cardiac haemodynamic data during the development of monocrotaline-induced pulmonary hypertension. Materials and methods All experiments were approved by the Institutional Animal Care and Use Committee of the VU University and were conducted in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, and the Dutch Animal Experimentation Act. Animals Thirty-eight male Wistar rats were used (250–300 g; Harlan, Horst, The Netherlands), of which 33 were operated and 5 served as controls (no surgery). The animals were allowed to adapt to their new environment for at least 1 week before surgery. The animals were conventionally housed in pairs under controlled conditions (temperature 21–22°C; humidity 60–65%; 12:12 h light–dark cycle) and had free access to filtered water and standard rat chow (Global 2016, Harlan Teklad, Blackthorn-Bicester, England). Telemetry system http://www.datasci.com/information/index.asp The pressure was monitored continuously during surgery to ensure proper position of the tip of the pressure catheter. After successful implantation of the transmitter, RV or PA pressures and locomotor activity were recorded for 10 s every 5 min. From the pressure recordings mean, systolic and diastolic pressure, heart rate, respiratory rate and circadian rhythm were subsequently derived (Dataquest A.R.T. software 4.0, DSI). Pre-operative care and anaesthesia 2 2 Animals were placed on a heating pad to maintain body temperature and positioned in dorsal recumbency. Hydromellose drops (0.3%; Ratiopharm, Zaandam, The Netherlands) were applied to prevent drying of the eyes. After shaving and disinfection with 70% ethanol of the chest and abdomen, animals were covered with sterile incision foil and the animals were then covered with sterile incision foil (Opraflex, Lohmann & Rauscher, Almere, The Netherlands). A surgical microscope was used (magnification ×16–64; Carl Zeiss, Sliedrecht, The Netherlands) for optimal view. Surgery: trans-thoracic approach Implantation of the telemetry transmitter in the abdomen The abdominal cavity was accessed via a 5-cm midline laparotomy, starting just below the xiphoid process. The transmitter was placed in the peritoneal cavity, with the catheter pointing caudally to prevent liver injury and tissue reaction. The abdominal cavity was covered with gauzes soaked in warm saline and left open until the end of the procedure. Routing of the pressure catheter to the right ventricle The heart was exposed by means of a left thoracotomy performed at the sixth intercostal space and mid-clavicular line. The thorax was opened with blunt scissors, respecting the anatomy of the overlying muscle layers and cautiously avoiding injury to the lungs. The pressure catheter was then tunnelled subcutaneously from the peritoneal cavity to the opening in the thorax and temporarily laid aside. Four individual small hooks were used to retract the ribs and expose the heart through a wider, 2 × 2-cm window. The pericardium was then opened with two dressing forceps. Catheterisation of the right ventricle 1 1 Fig. 1 left panel arrow right panel arrow  Closing of the chest and abdomen 2 Surgery: trans-diaphragm approach Opening of abdomen and the diaphragm 1 Catheterisation of the right ventricle RV catheterisation was performed similarly, as described above. In short, the pericardium was opened by two dressing forceps, and a purse string was placed just right of the apex. The pressure catheter was then inserted into the right ventricle through a small puncture made with a syringe needle and fixed in place. Closing the diaphragm and abdomen After catheterisation, blood clots in the thorax, if any, were removed. The diaphragm was closed with a running suture (5–0 Vicryl), starting from the ventral side, with the pressure catheter sticking out the diaphragm at the dorsal end of the incision. After that, the retraction suture through the xiphoid process was removed. To promote full expansion of the lungs, a small burst of positive pressure was applied, as described above, with the chest tube between the sutures of the diaphragm, until proper thorax excursions were visually confirmed. To further secure closure of the diaphragm, a small amount of tissue adhesive (Vetbond, 3M) was applied. Finally, the transmitter was implanted in the peritoneal cavity, fixed to the abdominal wall with the catheter facing caudally, and the abdomen was closed, as described above. Post-operative care To compensate for loss of fluids, all animals received warm sterile saline at the end of the procedure (5 ml intraperitoneal). After final skin closure, the animals were allowed to regain consciousness. In the first 24 h of recovery, the animals were housed in individual cages. Each cage was placed halfway on a heating pad, in such a way that half of the cage was maintained above it and the other at room temperature. During this period, the animals were monitored three times, received post-surgical analgesia when clinically indicated (buprenorphine 0.10 mg/kg subcutaneous) and were provided with drinking gel pads and softened rat chow. After 24 h, the animals were conventionally housed in pairs for a full recovery, and they were inspected and weighted daily. Animals were considered fully recovered from surgery when their appearance and behaviour were normal, their surgical wounds healed, their pre-surgical body weight regained, when the pressures normalised and circadian rhythm was restored. The study period was ended after 4 months. Experimental protocol 25 24 RV echocardiography 2 10 15 14 3 10 Estimation of pulmonary vascular resistance and RV power output Additional echocardiographic measurements were performed in MCT-treated rats and untreated but operated controls, just before the injection, 2 weeks after injection and when the first clinical signs of RV heart failure developed (as indicated by weight loss, dyspnoea and lethargy, after which the rats were euthanised). Pulmonary vascular resistance (PVR) and RV power output (RV-power) were estimated, by combining telemetric pressure data and echocardiographic flow data. 1 23 21 23 Autopsy 2 Statistical analyses t p Results Success rate Seventeen animals were operated using the trans-thoracic approach, and 16 animals were operated using the trans-diaphragm approach. In the trans-thoracic group, 8 animals showed stable pressure signals for the whole study period of 4 months. Five animals did not recover form surgery, and 4 animals developed instable pressure signals, caused by clot formation inside the pressure catheter within 2 weeks of recovery. Of the 9 failed procedures, 4 occured in the first four attempts. In the trans-diaphragm group, 9 animals showed stable pressure signal for the whole study period of 4 months. Six animals did not recover form surgery, and one animal developed instable pressure signals, caused by clot formation inside the pressure catheter within 2 weeks of recovery. As in the trans-thoracic group, 4 of the failures occurred in the first four attempts. By discarding the first 4 animals in each procedure, as we consider this the learning curve, the overall success rate was 8 of 13 (62%) for the trans-thoracic approach and 9 of 12 (75%) for the trans-diaphragm approach. Recovery from surgery 1 p Table 1 Main results of this study, all values in mean ± SEM   n n n a 8/13 (62%) 7/9 (79%) n.r. Recovery    d 9.5 ± 1.1 6.4 ± 0.5* n.r. e 362 ± 4.4 359 ± 5.7 b 8 17 e 90 ± 2.4 91 ± 3.8 b 11 17 20 e 3.4 ± 0.4 3.7 ± 0.6 n.r. e     RVSP (mmHg) 25 ± 1.1 24 ± 0.9 b 16  RVDP (mmHg) 1.7 ± 0.3 2.2 ± 0.2 c 5 d     Cardiac output (ml/min) 107 ± 4.9 116 ± 6.9 110 ± 4.9  TAPSE (mm) 3.6 ± 0.1 3.7 ± 0.2 3.4 ± 0.1  RVWT (mm) 1.0 ± 0.1 1.0 ± 0.1 0.9 ± 0.1  RVEDD (mm) 3.6 ± 0.1 3.7 ± 0.1 3.6 ± 0.1 f     Heart 1.4 ± 0.1 1.4 ± 0.2 1.3 ± 0.1  Lungs 1.5 ± 0.1 1.5 ± 0.2 1.5 ± 0.2  Liver 15.4 ± 0.5 15.0 ± 0.6 15.1 ± 0.5  Spleen 0.65 ± 0.03 0.68 ± 0.04 0.64 ± 0.04  Kidneys 2.4 ± 0.2 2.3 ± 0.2 2.3 ± 0.1 n n.r. BM RVSP RVDP TAPSE RVWT RVEDD p a b c d e f 1 1 1 Acute pressure effects of pulmonary embolisation by microspheres 1 p p Haemodynamic changes during the development of MCT-induced pulmonary hypertension 2 2 p p p p p Fig. 2 Triangles connected by dashed lines squares connected by uninterrupted lines n Asterisk p double asterisk p  9 −5 9 −5 9 −5 p p Autopsy 1 3 3 Discussion The present study describes in detail two surgical techniques to monitor RV or PA pressures over time by radio-telemetry in rats. To our best knowledge, we are the first research group to describe the trans-diaphragm approach and its comparison with the previously used trans-thoracic approach. We have demonstrated that: (1) in our hands, both the trans-diaphragm as well as the trans-thoracic approach have satisfactory success rates, especially when considering the complexity of the procedures, (2) the time-to-recovery was significantly shorter with the trans-diaphragm approach than the trans-thoracic approach, (3) measured physiological parameters recovered fully in both methods, and there were no permanent detrimental effects on RV, intercostal muscles or diaphragm, (4) using RV-telemetry, acute and chronic pressure changes in the pulmonary circulation can be readily detected and (5) RV-telemetry, in combination with echocardiography, allow thorough monitoring of pulmonary and cardiac haemodynamic changes over time. 4 7 2 26 In our experience, insertion of the pressure catheter into the RV and its advance further into the pulmonary artery when desired is comparatively easy to perform in the trans-diaphragm approach. This relative ease of RV catheterisation is reflected in the higher success rate compared to the trans-thoracic approach and might be well explained by the more caudal and inferior direction by which the catheter enters the heart. Approaching the heart in this manner entails minimal manipulation and manoeuvring of the catheter during the process of RV catheterisation, resulting in a lower incidence of clot formation inside the catheter. This complication occurred more frequently in trans-thoracic-operated animals as a direct consequence of excessive manoeuvring and accidental squeezing of the catheter causing it to lose or displace some of its anti-thrombolitic gel. The strongest advocate of the trans-diaphragm approach over the trans-thoracic approach, however, is the significantly faster recovery of the animals. Prompt recovery from surgery is particularly important in rats with MCT-induced PH because of low survival rates beyond 4 weeks after MCT injection. 23 18 In conclusion, we described a new, easier-to-perform, mildly invasive trans-diaphragm-based RV-telemetry approach for long-term monitoring of PA and RV pressures in the rat model of MCT-induced PH. Our findings may be applied to improve our understanding of the disease processes involved in PH and develop better treatment strategies for the disease. Electronic supplementary material Below is the link to the electronic supplementary material.
 Electronic Supplementary Material M.L. Handoko et al. A refined radio-telemetry technique to monitor right ventricle or pulmonary artery pressures in rats. Pflugers Arcg – Eur J Physiol 2007. Fig. E-1, E-2, E-3. (PDF 292 kb)