Neurodegeneration in the somatosensory cortex after experimental diffuse brain injury

Abstract

Disruption and consequent reorganization of central nervous system circuits following traumatic brain injury may manifest as functional deficits and behavioral morbidities. We previously reported axotomy and neuronal atrophy in the ventral basal (VB) complex of the thalamus, without gross degeneration after experimental diffuse brain injury in adult rats. Pathology in VB coincided with the development of late-onset aberrant behavioral responses to whisker stimulation, which lead to the current hypothesis that neurodegeneration after experimental diffuse brain injury includes the primary somatosensory barrel cortex (S1BF), which receives projection of VB neurons and mediates whisker somatosensation. Over 28 days after midline fluid percussion brain injury, argyrophilic reaction product within superficial layers and layer IV barrels at 1 day progresses into the cortex to subcortical white matter by 7 days, and selective inter-barrel septa and subcortical white matter labeling at 28 days. Cellular consequences were determined by stereological estimates of neuronal nuclear volumes and number. In all cortical layers, neuronal nuclear volumes significantly atrophied by 42-49% at 7 days compared to sham, which marginally attenuated by 28 days. Concomitantly, the number of healthy neurons was reduced by 34-45% at 7 days compared to sham, returning to control levels by 28 days. Progressive neurodegeneration, including argyrophilic reaction product and neuronal nuclear atrophy, indicates injury-induced damage and reorganization of the reciprocal thalamocortical projections that mediate whisker somatosensation. The rodent whisker barrel circuit may serve as a discrete model to evaluate the causes and consequences of circuit reorganization after diffuse brain injury.

Fig. 1

Diffuse brain injury modeled by midline fluid percussion injury (FPI) occurs in the absence of overt hemorrhage, edema or cavitation. Distinct cortical layers based on cytology remain preserved in primary somatosensory barrel cortex (layers I–VI) over time post-injury. Cortical thickness was not measured. Scale Bar = 100 μm

Fig. 2

Post-traumatic time course of argyrophilic reaction product in the primary somatosensory barrel cortex as revealed by the de Olmos silver staining technique in uninjured sham (a) and at 1 day (b), 7 days (c) and 28 days (d) after moderate midline fluid percussion brain injury. Uninjured sham brain was absent of argyrophilic reaction product. At 1 day post-injury, argyrophilic reaction product was localized to the superficial segment of the cortex, particularly within the cortex barrels (between white arrowheads). At 7 days post-injury, argyrophilic reaction product progressed to the middle segment of the cortex and remained preferentially in the cortical barrels. By 28 days post-injury, argyrophilic reaction product appears absent in the superficial segment and diffuse across the middle and deeper segments of the cortex. By this time, staining encompasses the inter-barrel septa (white arrowheads)

Fig. 3

Argyrophilic reaction product progresses through the cortex over time post-injury. a Photomicrographs of primary somatosensory barrel cortex (S1BF) processed by the de Olmos silver staining technique show no appreciable accumulation in uninjured sham brain and indication of degeneration that progresses to deeper segments of the cortex with time post-injury. The cortical mantle has been divided by thirds into superficial, middle and deep segments. b Higher power photomicrographs of each segment of S1BF cortex show the progression through deeper segments and dense argyrophilic reaction product in neuronal processes, which likely include axons, dendrites and synaptic terminals. Infrequent stained somata are present, primarily at 1 day post-injury in the middle and deep segments. c Argyrophilic reaction product was quantified as a percent of silver stained pixels in each cortical segment (pixel quantification; see “Materials and methods”). As visualized, degeneration in the superficial segment increased significantly at 1 day after fluid percussion injury (FPI) and subsided over 28 days. In the middle segment, degeneration continued to increase over 1 and 7 days post-injury and remained elevated at 28 days post-injury compared to uninjured sham. In the deep segment, a delayed increase in degeneration was observed at 7 and 28 days post-injury compared to sham. (mean ± SEM; *p <0.05 compared to sham; ⁺p <0.05 compared to 1 day FPI; *p <0.05 compared to 7 days FPI)

Fig. 4

Brain injury-induced atrophy of neuronal nuclear volume was estimated using the nucleator probe. Fluid percussion injury (FPI) results in a significant reduction in mean neuronal nuclear volume in all cortical layers at 7 days post-injury compared to sham (*p <0.05). Injury-induced neuronal atrophy is evident in Layer V at all post-injury time points compared to sham (*p <0.05). Values are mean ± SEM

Fig. 5

Injury-induced shifts in neuronal nuclear size distribution across cortical layers in the primary somatosensory barrel fields. Nucleator measurements were pooled across sections and binned by size. The frequency of each volume measurement is plotted for sham and fluid percussion injured (FPI) animals. After brain injury, the nuclear size distributions show significant shifts toward smaller volumes (mean ± SEM; * and ⁺p <0.05 compared to sham and 7 days FPI for each size bin, respectively). Neuronal populations from brain-injured animals show fewer neurons that are larger than the mean of the sham group (grey vertical line). Additionally, for brain-injured animals, there are more neurons in the bins smaller than the mean of the sham group

Fig. 6

Stereological quantification in the S1BF using the fractionator and Cavalieri principles to estimate neuronal number and regional volume, respectively. a At 7 days post-injury, the number of neurons in Layer II/III was significantly less than sham and other post-injury time points. Neuronal number is significantly different between 7 and 28 days post-injury in layer V. b No injury-related changes in regional volume were detected. Estimates of regional volume were obtained by the Cavalieri method, based on systematically random point counting. c Neuronal density calculations indicate significant injury-related reductions at 7 days post-injury compared to sham and other post-injury time points. (mean ± SEM; *p <0.05 compared to sham; ⁺p <0.05 compared to 7 days FPI)