A method to assess invasion and intracellular replication of Trypanosoma cruzi based on differential uracil incorporation

The retrograde tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label sympathetic preganglionic neurons (SPN) and motoneurons (MN) in postmortem human spinal cord. Seven months after microinjection of DiI into the ventral part of spinal thoracic segments T4 and T8, DiI-labelled neurons were identified and analyzed. Cryostat sections of spinal cord were prepared for light microscopy, while vibratome sections were analyzed using confocal microscopy. The majority of retrogradely labelled SPNs were located within the intermediolateral nucleus, with a few labelled dendrites having a mediolateral orientation. SPNs were also located within the nucleus intercalatus, around the central canal and in the lateral funiculus. Cell bodies of retrogradely labelled IML neurons were oval, kite- or spindle-shaped. The soma area of SPNs in T4 was approximately 422.9 +/- 20.9 microm2 with a median diameter of 14 +/- 0.6 microm. MNs in the ventral horn were round or oval in shape and often appeared with a few labelled neurites. The soma area of the MNs in T4 was approximately 842.3 +/- 35.1 microm2, with a median diameter of 18.3 +/- 0.1 microm. The mean values for MN soma area and diameter measurements were significantly greater compared to SPNs. However, no difference was observed between MNs in different segments or between SPNs in the same segments. No retrogradely labelled cells were observed within the dorsal horn. These findings indicate that DiI is a useful method for studying fixed human central nervous system tissue.