Turbina de impulso axial con aletas directrices fijas 57

monitoring diseases. Similar procedures were, therefore, required to be developed in experimental animal models to simulate the clinical situations for studying the unknown mechanism of disorders. Considering the sensitivity of the method used for detecting specific CSF elements and the available volume of CSF, most of the repeated sampling CSF techniques were previously developed in fairly big animals, such as non-human primates, dogs, and cats. With the advantage of more sensitive detecting methods, the application of similar methods to the much smaller volumes of CSF available from small animals has been possible (Sarna, et al., 1983). Repeated sampling of as small a quantity as a few microlitres from the conscious rat offers a method for experimentally studying cerebral metabolism in the small animal and thus repeatedly monitoring changes in specific elements of the brain. However, in my experience, the previously reported method (Sama, et al., 1983) had several obvious disadvantages. The first is a low long-term patency rate (only a few percent), which makes the method unsuitable for chronic experiments lasting for weeks. Secondly, the cannula has to be inserted into the cisterna magna blindly using a metal wire pushed

C hapter!. Experim ental m ethods

down inside. This may damage the surface vessels of the brain and cause intracranial bleeding. In addition, the cannula has to travel subdurally all the way from the vertex to the cisterna magna thus occupying a large volume of the intracranial space which may be crucial for animal survival if cerebral oedema is induced afterwards. I therefore modified the method by implanting a polyethylene tube in the rat cisterna magna via an opened occipital transcranial approach in order to obtain a better view of the cistern to avoid damage to the surface vessels during implantation. The intracranial segment of the approach, leading to the cisterna magna, was only a few millimetres in length and the intracranial space occupied by the implanted cannula was thus minimised. Accurate placement of the cannula into the subarachnoid space increased its long-term patency rate so that the cannula became suitable for experiments lasting for weeks.

The surgical details are as follows: The cannula (with spare ones), surgical instruments and other materials including glass container and pipettes for mixing dental acrylic

were sterilized by either 15 min steaming in a boiler or heating in an oven at 100°C overnight (standard procedure for sterile surgery). The animal was anaesthetized by injecting Sagatal (60 mg/kg, i.p.) and placed in a small-animal stereotaxic frame. A midline incision was made between the lambda and the external occipital crest. Skin and underlying tissues were retracted to expose the dorsal skull, and a burr hole was drilled for the placement of a stainless steel screw (which was also used as a gross superficial electrode for EP recording) for securing the implanted cannula later on. The middle part of the occipital muscles were dissected from the skull down to the upper bony edge of the foramen magnum, and retracted caudally to expose the back of

C hapter!. E xperim ental m ethods

the skull. Under a low-power surgical microscope, another bigger burr hole ( 1 x 2 mm) was made in the midline, 2 mm from the bony edge of the foramen magnum, with care being taken to keep the dura intact. The subarachnoid space was opened with the tip of a sharp 30G needle. A ‘J - shape’ 6 - 8 cm PE 10 polyethylene tube (Portex Ltd, Kent) held by a manipulator was implanted into the cisterna magna. Correct placement of the cannula was indicated by the ability to withdrawing the CSF gently via the cannula without any difficulty. After surrounding area had been dried, the cannula and stainless steel screw were secured in place with dental acrylic (Austenal Dental Products Ltd, Harrow), and a few stitches finally inserted as necessary around the incision. After a short period of training, one could expect to complete such surgery within 30-40 minutes, during which adequate surgical anaesthesia was maintained by original single i.p. injection of Sagatal. The tail of the catheter was then cut to a length of approximately 4 cm, and its free end covered by a 0.5 cm long, heat- sealed PESO tube (Fig. 2-1). After 2 days’ recovery, the animals invariably appeared to be in good health. A few more samples of 50|xl were taken by a small syringe with 30G needle inserted into the PE 10 cannula within the next a few days to clear any CSF possibly contaminated by the surgical procedure.

Further repeated samplings of 50pl were achieved via the cannula by connecting a 30G needle and a 50pl syringe at the same time as the AEP was recorded. A volume of CSF sample of 50pl (approximate 10% of total CSF volume in a rat) was considered as the maximal practical volume that could be withdrawn safely without significantly affecting the physiological condition of the CSF circulation or causing bleeding from the superficial vessels of the brain due to a change in the intracranial

C hapter!. E xperim ental m ethods I— PESO — PE10 CB OS w J L # w v m m v m . u w m a a j l a v a a m A A m ^ v r «^v/JL h a m a a a u^ a a m

repeated CSF sampling in rats. *: cisterna magna; BS: