Neuronal and glial changes in the rat phrenic nucleus occurring within hours after spinal cord injury

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Abstract
The present study describes specific morphological changes in the normal ultrastructure of the rat phrenic nucleus which occur within 4 hours after an ipsilateral spinal cord hemisection rostral to the nucleus. Phrenic neurons were identified at EM levels by retrograde HRP labeling. Ultrastructural features of the phrenic nucleus in uninjured animals and at 4 hours and 1, 2, and 4 days after injury were qualitatively analyzed and then quantitated with a computerized morphometric system. Our results indicated that by 4 hours posthemisection, there was a significant increase in the number of double synapses. Furthermore, the number of double synapses remained significantly higher than normal at all the other posthemisection periods. A significant increase in the length of dendrodendritic membrane appositions was also noted as early as 4 hours posthemisection. The mean normal appositional length of 1.42 ± 0.09 μm increased to 1.89 ± 0.12 μm at 4 hours and further increased to 2.20 ± 0.20 μm by 1 day posthemisection. The increase in the length of membrane appositions was most likely due to an active retraction of astroglial processes from their normal position in between the dendrites. Although there was an increase in the mean length of the dendrodendritic appositions, the mean percentage of the appositions (expressed as the total number of appositions divided by the total number of dendrites in each sample) was not increased significantly over normal values during the early posthemisection periods. By 2 and 4 days posthemisection, however, the percentage of dendrodendritic appositions increased to significantly higher values than normal. Normally, 4.68 ± 0.69% of the dendrites in the phrenic nucleus were found to be in apposition, and this number increased significantly to 7.27 ± 1.06% by 2 days and 7.46 ± 0.79% by 4 days posthemisection. At these later posthemisection periods, the mean length of the appositions decreased to levels which were no longer significantly higher than normal. A distribution analysis of the length of each dendrodendritic apposition in both the normal and spinal hemisected rats showed that there were more dendrodendritic appositions in the phrenic nucleus at the later posthemisection periods. It also showed that their mean length was decreased because many of the new appositions were relatively short. The above neuronal and glial alterations of the phrenic nucleus have never before been described as a response to injury of the mammalian spinal cord. Furthermore, the possibility that the above changes could represent the morphological substrate for the unmasking of functionally ineffective synapses in our spinal cord injury model is discussed.