Improved immunocytochemical identification of neural, endothelial, and inflammatory cell types in paraffin-embedded injured adult rat spinal cord

Methods that facilitate the accurate counting of specific neural cell types would be of substantial value in evaluating the efficacy of treatments applied to spinal cord injury. This report describes reliable procedures for identification of neurons, oligodendrocytes, astrocytes, endothelial cells and inflammatory cells (neutrophils and activated macrophage/microglial cells) in paraformaldehyde-fixed, paraffin-embedded injured adult rat spinal cord. Antigen retrieval techniques (enzymatic and thermal) were used to improve antibody access to masked epitopes. To decrease background immunofluorescence and autofluorescence of hemoglobin, the tissue sections were pretreated with 0.1% sodium borohydride in PBS (30 min), followed by 1–5 min incubation in 0.5% Sudan black in 70% ethanol. Commercially available techniques to amplify the primary signal such as tyramide signal amplification (TSA) and avidin/biotin/peroxidase/DAB/nickel/cobalt amplification (ABP/DABA) were also tested. Hoechst 33342 nuclear staining was used to indicate cell location, number, and integrity, thereby avoiding misidentification of cells. The best antibodies were: anti-NeuN antibody for neurons, anti-S100 for astrocytes, and anti-S100 and APC-7 antibodies in combination for oligodendrocytes, anti-laminin (LN) for endothelial cells, and ED1 antibody for activated macrophages and microglia. Amplification of the primary signal with TSA or ABP/DABA was also found to be beneficial

Endothelial cell loss is not a major cause of neuronal and glial cell death following contusion injury of the spinal cord

Contusion of the spinal cord causes an immediate local loss of neurons and disruption of vasculature; additional loss continues thereafter. To explore the possibility of a causal link between delayed endothelial cell (EC) death and secondary neural cell loss, we evaluated neural and endothelial cell survival, and measured inflammatory cell infiltration, at times up to 48 h after contusion injury to the adult rat thoracic spinal cord. Female Fischer rats (200 g), subjected to moderate (10 g × 12.5 mm) weight drop injuries by the MASCIS (NYU) impactor, were analyzed at 15 min and at 1, 8, 24 and 48 h. ECs, neurons, astrocytes, oligodendrocytes, neutrophils and activated macrophages/microglia were counted in transverse sections. At the injury site, 90% of all neurons died within 48 h of injury; no medium–large diameter neurons survived beyond 48 h. EC death occurred with kinetics similar to glial cell death. Because, in the injury site, most cell death occurred before 8 h, it preceded inflammatory cell infiltration. Three millimeters rostral and caudal to the injury epicenter neuronal numbers were stable for 8 h, and a sharp decrease in neuronal numbers beginning at 8 h strongly correlated with the onset of inflammatory cell infiltration. Glial and blood vessel numbers remained relatively stable in these areas up to 48 h. These results suggest that the loss of ECs during the first 48 h after a contusion injury is not a major cause of neuronal and glial cell death and, in tissue adjacent to the epicenter, inflammatory cell infiltration leads to neuronal loss

New Vascular Tissue Rapidly Replaces Neural Parenchyma and Vessels Destroyed by a Contusion Injury to the Rat Spinal Cord

Blood vessels identified by laminin staining were studied in uninjured spinal cord and at 2, 4, 7, and 14 days following a moderate contusion (weight drop) injury. At 2 days after injury most blood vessels had been destroyed in the lesion epicenter; neurons and astrocytes were also absent, and few ED1+ cells were seen infiltrating the lesion center. By 4 days, laminin associated with vessel staining was increased and ED1+ cells appeared to be more numerous in the lesion. By 7 days after injury, the new vessels formed a continuous cordon oriented longitudinally through the lesion center. ED1+ cells were abundant at this time point and were found in the same area as the newly formed vessels. Astrocyte migration from the margins of the lesion into the new cordon was apparent. By 14 days, a decrease in the number of vessels in the lesion center was observed; in contrast, astrocytes were more prominent in those areas. In addition to providing a blood supply to the lesion site, protecting the demise of the newly formed vascular bridge might provide an early scaffold to hasten axonal regeneration across the injury site

Motoneuron Replacement for Reinnervation of Skeletal Muscle in Adult Rats

Reinnervation is needed to rescue muscle when motoneurons die in disease or injury. Embryonic ventral spinal cord cells transplanted into peripheral nerve reinnervate muscle and reduce atrophy but low motoneuron survival may limit motor unit formation. We tested whether transplantation of a purified population of embryonic motoneurons into peripheral nerve (mean ± SE: 146,458 ± 4011 motoneurons) resulted in more motor units and reinnervation than transplantation of a mixed population of ventral spinal cord cells (72,075 ± 12,329 motoneurons). Ten weeks after either kind of transplant, similar numbers of neurons expressed choline acetyl transferase and/or Islet-1. Motoneuron numbers always exceeded the reinnervated motor unit count. Most motor end plates were simple plaques. Reinnervation significantly reduced muscle fiber atrophy. These data show that the number of transplanted motoneurons or motoneuron survival do not limit muscle reinnervation. Incomplete differentiation of motoneurons in nerve and lack of muscle activity may result in immature neuromuscular junctions that limit reinnervation and function