For histological studies, twenty one rats received a 400 g/cm focal trauma to the SMCX using the weight-drop-method (3) under halothane anesthesia and later anesthetized with sodium pentobarbital, transcardially perfused with 0.9% physiological saline followed by 10% formalin at 6 hr (n=4), 24 hr (n=4), 48 hr (n=4), 72 hr (n=3) and 25-30 days (n=6). Serial frozen 40 micron sections were cut using a cryostat and stained using routine procedures.
To confidently determine the cortical area following SMCX, destined to evolve into a cyst, the overlapping areas of the cyst for six animals, killed at 25-30 days post injury (when volume expansion has ceased), was drawn at the center plane of impact (-2.56 mm from bregma; Paxinos and Watson, 1986). A sampling grid (3 mm x 3 mm) was then drawn within this small area to assess the percentage of viable or injured neurons within each sector and at early time points following SMCX contusion, prior to cyst development. As illustrated in Figure 1 this grid was divided into three horizontal sectors: upper (cortical layers I and II), middle (cortical layers III and IV), and lower (cortical layers V and VI) lamina. The grid was then further divided into three lateral sectors Each sector was 1 mm wide.
To assess the viability of neurons in the cortical area destined to evolve into a cyst, sections from animals sacrificed early after injury were stained using three stains, each frequently utilized as a marker of severe neuronal injury or pending cell death. These stains included:
Eleven rats were prepared for study of regional cerebral blood flow (rCBF) with the quantitative Iodo-14C-antipyrine autoradiographic technique at 24 hours after the 400 g/cm 400 g/cm SMCX contusion. Analysis of rCBF was quantified by microdensitometry of the autoradiograms and the percentages reported are of the contralateral homologous area.
Lastly, using an image analysis system , Khoros, developed at The University of New Mexico, the serial sections from one rat were reconstructed to depict the 3-dimensional shape of the posttraumatic cortical cyst.