Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat.

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC-ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100x1 mm I.D. C8, 5 microm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 microl/min. For monoamines and metabolites we used a 150X1 mm I.D. C18 5 microm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 microM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-microm tissue slices. Tissue samples were homogenized in 50 or 100 microl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and gamma-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.

Some functional recovery and behavioral sparing occurs independent of task-specific practice after injury to the rat's sensorimotor cortex.

These experiments on rats evaluated whether recovery of competence in certain motor tests could be enhanced by practice begun soon after traumatic brain injury (TBI). Before TBI, rats were pre-trained to cross a flat and a pegged beam. Anesthetized animals received a right sensorimotor cortex TBI. One group began task-specific testing (flat and pegged beams) on day 1 after injury and repeated 13 times in 35 days by which time functional recovery occurred. Paw preference was evaluated eight times during the 35 day period, beginning the third day after injury. A second group of injured rats remained in their home cage without any testing for 35 days after injury. From day 35 they were tested 13 times over the next 35 days on both beam tests and eight times on the paw preference test. At day 35 those rats that remained in their home cage without testing (task-specific practice) performed as well on the flat beam as the rats that began testing 1 day after injury. By day 37, their third test day, the untested rats performed as well as the tested rats on the pegged beam. Paw preference was the same in both groups of rats. These results were compared to sham-operated controls. Post-injury performance as measured by these tests indicated that most of the recovery occurred without task-specific practice. However, task-specific practice was necessary to achieve optimum performance on both beam tests. This implies that neural reorganization occurred independent of any practice. Task specific practice served to 'fine tune' the rat's performance after 35 days.

Ladder beam and camera video recording system for evaluating forelimb and hindlimb deficits after sensorimotor cortex injury in rats.

Hindlimb and forelimb deficits in rats caused by sensorimotor cortex lesions are frequently tested by using the narrow flat beam (hindlimb), the narrow pegged beam (hindlimb and forelimb) or the grid-walking (forelimb) tests. Although these are excellent tests, the narrow flat beam generates non-parametric data so that using more powerful parametric statistical analyses are prohibited. All these tests can be difficult to score if the rat is moving rapidly. Foot misplacements, especially on the grid-walking test, are indicative of an ongoing deficit, but have not been reliably and accurately described and quantified previously. In this paper we present an easy to construct and use horizontal ladder-beam with a camera system on rails which can be used to evaluate both hindlimb and forelimb deficits in a single test. By slow motion videotape playback we were able to quantify and demonstrate foot misplacements which go beyond the recovery period usually seen using more conventional measures (i.e. footslips and footfaults). This convenient system provides a rapid and reliable method for recording and evaluating rat performance on any type of beam and may be useful for measuring sensorimotor recovery following brain injury.