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Differential Histopathological and Behavioral Outcomes Eight Weeks after Rat Spinal Cord Injury by Contusion, Dislocation, and Distraction Mechanisms

View Article: PubMed Central - PubMed

ABSTRACT

The objective of this study was to compare the long-term histological and behavioral outcomes after spinal cord injury (SCI) induced by one of three distinct biomechanical mechanisms: dislocation, contusion, and distraction. Thirty male Sprague-Dawley rats were randomized to incur a traumatic cervical SCI by one of these three clinically relevant mechanisms. The injured cervical spines were surgically stabilized, and motor function was assessed for the following 8 weeks. The spinal cords were then harvested for histologic analysis. Quantification of white matter sparing using Luxol fast blue staining revealed that dislocation injury caused the greatest overall loss of white matter, both laterally and along the rostrocaudal axis of the injured cord. Distraction caused enlarged extracellular spaces and structural alteration in the white matter but spared the most myelinated axons overall. Contusion caused the most severe loss of myelinated axons in the dorsal white matter. Immunohistochemistry for the neuronal marker NeuN combined with Fluoro Nissl revealed that the dislocation mechanism resulted in the greatest neuronal cell losses in both the ventral and dorsal horns. After the distraction injury mechanism, animals displayed no recovery of grip strength over time, in contrast to the animals subjected to contusion or dislocation injuries. After the dislocation injury mechanism, animals displayed no improvement in the grooming test, in contrast to the animals subjected to contusion or distraction injuries. These data indicate that different SCI mechanisms result in distinct patterns of histopathology and behavioral recovery. Understanding this heterogeneity may be important for the future development of therapeutic interventions that target specific neuropathology after SCI.

No MeSH data available.


Histological regions of interest. (a) To measure area of spared tissue in Luxol fast blue (LFB)-stained sections, the dorsal, lateral, and ventral white matter and the gray matter were segmented. The lateral white matter was separated from the dorsal and ventral white matter by drawing straight lines along the lateral sides of the dorsal and ventral horns. (b) To calculate density of myelinated axons in neurofilament H/β-tubulin/myelin basic protein-stained sections, several boxes (65 μm × 65 μm) were placed. One box was positioned in the center of the gracile fasciculus. Another box was positioned in the middle right of the dorsal corticospinal tract. A third box was positioned in the right cuneate fasciculus 50 μm away from the gracile fasciculus and the dorsal corticospinal tract. A fourth box (100 μm × 100 μm) was positioned in the right lateral white matter next to the dorsal horn. One box (100 μm wide) was positioned in the right ventral white matter medial from the ventral nerve root. The size and position of the boxes could be adjusted to avoid large artifacts and horizontal axons. (c) To count the number of surviving cells in NeuN/Fluoro Nissl-stained sections, images were taken in the right dorsal horn (20x) and the right ventral horn (10x) with their sides aligned with the edges of the horns.
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f2: Histological regions of interest. (a) To measure area of spared tissue in Luxol fast blue (LFB)-stained sections, the dorsal, lateral, and ventral white matter and the gray matter were segmented. The lateral white matter was separated from the dorsal and ventral white matter by drawing straight lines along the lateral sides of the dorsal and ventral horns. (b) To calculate density of myelinated axons in neurofilament H/β-tubulin/myelin basic protein-stained sections, several boxes (65 μm × 65 μm) were placed. One box was positioned in the center of the gracile fasciculus. Another box was positioned in the middle right of the dorsal corticospinal tract. A third box was positioned in the right cuneate fasciculus 50 μm away from the gracile fasciculus and the dorsal corticospinal tract. A fourth box (100 μm × 100 μm) was positioned in the right lateral white matter next to the dorsal horn. One box (100 μm wide) was positioned in the right ventral white matter medial from the ventral nerve root. The size and position of the boxes could be adjusted to avoid large artifacts and horizontal axons. (c) To count the number of surviving cells in NeuN/Fluoro Nissl-stained sections, images were taken in the right dorsal horn (20x) and the right ventral horn (10x) with their sides aligned with the edges of the horns.

Mentions: The cross-sectional area of spared tissue (blue-labeled pixels that were darker than the background, representing myelin), the density of myelinated axons (NF/Tub/MBP staining), and the number of surviving cells (NeuN/Fluoro Nissl staining) were measured in the regions of interest (ROIs) in these sections (Fig. 2). In addition, the cross-sectional area of spinal cords, including the anterior median fissure and central canal, was outlined and measured from the LFB-stained sections. The central lesion cavity in the LFB-stained sections was also outlined to calculate the volume and rostrocaudal extent of the lesion. Sections at certain distances from the epicenter were selected from each set of spinal cords for analysis (see Fig. 5, 6, 9 for the specific locations).


Differential Histopathological and Behavioral Outcomes Eight Weeks after Rat Spinal Cord Injury by Contusion, Dislocation, and Distraction Mechanisms
Histological regions of interest. (a) To measure area of spared tissue in Luxol fast blue (LFB)-stained sections, the dorsal, lateral, and ventral white matter and the gray matter were segmented. The lateral white matter was separated from the dorsal and ventral white matter by drawing straight lines along the lateral sides of the dorsal and ventral horns. (b) To calculate density of myelinated axons in neurofilament H/β-tubulin/myelin basic protein-stained sections, several boxes (65 μm × 65 μm) were placed. One box was positioned in the center of the gracile fasciculus. Another box was positioned in the middle right of the dorsal corticospinal tract. A third box was positioned in the right cuneate fasciculus 50 μm away from the gracile fasciculus and the dorsal corticospinal tract. A fourth box (100 μm × 100 μm) was positioned in the right lateral white matter next to the dorsal horn. One box (100 μm wide) was positioned in the right ventral white matter medial from the ventral nerve root. The size and position of the boxes could be adjusted to avoid large artifacts and horizontal axons. (c) To count the number of surviving cells in NeuN/Fluoro Nissl-stained sections, images were taken in the right dorsal horn (20x) and the right ventral horn (10x) with their sides aligned with the edges of the horns.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035937&req=5

f2: Histological regions of interest. (a) To measure area of spared tissue in Luxol fast blue (LFB)-stained sections, the dorsal, lateral, and ventral white matter and the gray matter were segmented. The lateral white matter was separated from the dorsal and ventral white matter by drawing straight lines along the lateral sides of the dorsal and ventral horns. (b) To calculate density of myelinated axons in neurofilament H/β-tubulin/myelin basic protein-stained sections, several boxes (65 μm × 65 μm) were placed. One box was positioned in the center of the gracile fasciculus. Another box was positioned in the middle right of the dorsal corticospinal tract. A third box was positioned in the right cuneate fasciculus 50 μm away from the gracile fasciculus and the dorsal corticospinal tract. A fourth box (100 μm × 100 μm) was positioned in the right lateral white matter next to the dorsal horn. One box (100 μm wide) was positioned in the right ventral white matter medial from the ventral nerve root. The size and position of the boxes could be adjusted to avoid large artifacts and horizontal axons. (c) To count the number of surviving cells in NeuN/Fluoro Nissl-stained sections, images were taken in the right dorsal horn (20x) and the right ventral horn (10x) with their sides aligned with the edges of the horns.
Mentions: The cross-sectional area of spared tissue (blue-labeled pixels that were darker than the background, representing myelin), the density of myelinated axons (NF/Tub/MBP staining), and the number of surviving cells (NeuN/Fluoro Nissl staining) were measured in the regions of interest (ROIs) in these sections (Fig. 2). In addition, the cross-sectional area of spinal cords, including the anterior median fissure and central canal, was outlined and measured from the LFB-stained sections. The central lesion cavity in the LFB-stained sections was also outlined to calculate the volume and rostrocaudal extent of the lesion. Sections at certain distances from the epicenter were selected from each set of spinal cords for analysis (see Fig. 5, 6, 9 for the specific locations).

View Article: PubMed Central - PubMed

ABSTRACT

The objective of this study was to compare the long-term histological and behavioral outcomes after spinal cord injury (SCI) induced by one of three distinct biomechanical mechanisms: dislocation, contusion, and distraction. Thirty male Sprague-Dawley rats were randomized to incur a traumatic cervical SCI by one of these three clinically relevant mechanisms. The injured cervical spines were surgically stabilized, and motor function was assessed for the following 8 weeks. The spinal cords were then harvested for histologic analysis. Quantification of white matter sparing using Luxol fast blue staining revealed that dislocation injury caused the greatest overall loss of white matter, both laterally and along the rostrocaudal axis of the injured cord. Distraction caused enlarged extracellular spaces and structural alteration in the white matter but spared the most myelinated axons overall. Contusion caused the most severe loss of myelinated axons in the dorsal white matter. Immunohistochemistry for the neuronal marker NeuN combined with Fluoro Nissl revealed that the dislocation mechanism resulted in the greatest neuronal cell losses in both the ventral and dorsal horns. After the distraction injury mechanism, animals displayed no recovery of grip strength over time, in contrast to the animals subjected to contusion or dislocation injuries. After the dislocation injury mechanism, animals displayed no improvement in the grooming test, in contrast to the animals subjected to contusion or distraction injuries. These data indicate that different SCI mechanisms result in distinct patterns of histopathology and behavioral recovery. Understanding this heterogeneity may be important for the future development of therapeutic interventions that target specific neuropathology after SCI.

No MeSH data available.