Background: Two-photon laser scanning microscopy (TPLSM) currently provides the most powerful tool for fluorescence imaging of living tissues in vitro and in vivo. TPLSM measurements rely on extremely high stability of the tissue. In vivo this has only been achieved so far by fixation of the skull for brain imaging. In the spinal cord nociceptive afferences project to lamina I projection neurons which play an important role in chronic pain. Spinal cord slices lack of modulatory descending input and no natural noxious stimuli can be applied. Therefore, there was a demand for a stable in vivo model to study pain pathways. In this study we developed a rat model of cardiopulmonary bypass (CPB) using a heart-lung-machine as a form of anaesthesia that provides sufficient mechanical stability for in vivo TPLSM Ca2+-imaging in the spinal cord. Methods: Male Sprague Dawley rats (80-120 g) were anaesthetised with isoflurane and mechanically ventilated. The femoral artery was cannulated for continuous blood pressure recording and periodical arterial blood gas measurements. The heart frequency was monitored by subcutaneously placed ECG electrodes.
The Nervus ischadicus was prepared to apply electrical stimuly in C-fiber strength. A laminectomy was performed for lumbal spinal cord segments (L4/L5) for TPLSM access. Dorsal horn neurons were labelled with Oregon Green 488 BAPTA-1 AM. Results: The extracorporal circuit was composed of a venous reservoir, a membrane oxygenator, a heat exchanger and a roller pump. CPB was conducted for 3 hours at a flow rate of 150 ml/kg/min. Haemodynamic examinations and arterial blood gas analyses displayed animals within physiological conditions. Subsequent to the experiment blood chemistry profiles were established, rats underwent an autopsy and pathohistology was done from the brain and the spinal cord.
Two out of six CPB rats showed typical morphological changes of ischemia-related cell death in the hippocampal CA1 region. Conclusions:
This rat model of CPB provides mechanical stability for in vivo TPLSM Ca2+ Imaging in lamina I neuronal somata, axons and dendrites.
rat cardiopulmonary bypass / in vivo / rat / two photon laser scanning microscopy / pain / calcium imaging / long term potentiation / extracorporeal circulation