Neuroprotective effects of perflurocarbon (oxycyte) after contusive spinal cord injury

Abstract

Spinal cord injury (SCI) often results in irreversible and permanent neurological deficits and long-term disability. Vasospasm, hemorrhage, and loss of microvessels create an ischemic environment at the site of contusive or compressive SCI and initiate the secondary injury cascades leading to progressive tissue damage and severely decreased functional outcome. Although the initial mechanical destructive events cannot be reversed, secondary injury damage occurs over several hours to weeks, a time frame during which therapeutic intervention could be achieved. One essential component of secondary injury cascade is the reduction in spinal cord blood flow with resultant decrease in oxygen delivery. Our group has recently shown that administration of fluorocarbon (Oxycyte) significantly increased parenchymal tissue oxygen levels during the usual postinjury hypoxic phase, and fluorocarbon has been shown to be effective in stroke and head injury. In the current study, we assessed the beneficial effects of Oxycyte after a moderate-to-severe contusion SCI was simulated in adult Long-Evans hooded rats. Histopathology and immunohistochemical analysis showed that the administration of 5 mL/kg of Oxycyte perfluorocarbon (60% emulsion) after SCI dramatically reduced destruction of spinal cord anatomy and resulted in a marked decrease of lesion area, less cell death, and greater white matter sparing at 7 and 42 days postinjury. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining showed a significant reduced number of apoptotic cells in Oxycyte-treated animals, compared to the saline group. Collectively, these results demonstrate the potential neuroprotective effect of Oxycyte treatment after SCI, and its beneficial effects may be, in part, a result of reducing apoptotic cell death and tissue sparing. Further studies to determine the most efficacious Oxycyte dose and its mechanisms of protection are warranted.

FIG. 1.

Oxycyte preserves myelin and white matter after SCI. Light microscopic analysis Luxol Fast Blue/cresyl violet–stained sections 1 mm caudal to the lesion epicenter 7 days after impaction (a–k). Panel 1: animal group sustained laminectomy; 2: animals sustained contusion; 3: animal group sustained contusion and given saline (5 mL/kg); 4: animal group sustained contusion and given Oxycyte (5 mL/kg). Representative micrographs (b, e, h, and k) 20× magnified area corresponding to 2.5× micrograph of spinal cord area indicated by arrowhead in sections (a–j). Note that myelin tracts (blue or green area) within the lesion are better preserved in Oxycyte-treated animals, whereas they are mostly lost in the saline group. Micrographs of hematoxylin and eosin–stained sections (c, f, i, and l) correspond to the different levels of cellular damage observed between the groups. Staining shows an extensive tissue and neuron damage, as well as hypertrophic cellular debris in contused untreated animals (f), whereas Oxycyte animals (l) have better-preserved tissues and neurons (fewer vacuoles [V] and better-preserved Nissl [N]), compared to tissular spongiosis and retracted Nissl in the saline group (i). Cresyl violet staining (40×) of motor neuronal cell bodies analyzed 7 days postcontusion shows large numbers of foamy macrophages and cellular debris (arrow head) in spinal cord sections of contused animal panel 2 (n) and injured Nissl in saline-injured animals (o), but better-preserved neurons in Oxycyte-injured animals, whereas laminectomy alone showed normal motor neuronal cell bodies. SCI, spinal cord injury. Color image is available online at www.liebertpub.com/neu

FIG. 2.

Oxycyte reduces lesion size and spares white matter after SCI. Light microscopic analysis, Luxol Fast Blue/cresyl violet staining of representative spinal cord cross-sections showing the different lesion size for the different treatment groups (saline- and Oxycyte-injured animals). The photomicrographs of Luxol Fast Blue–stained sections 24 h postcontusion show damage to the white and gray matter and loss of myelin in all spinal cord sections after SCI, whereas laminectomy sections have no evidence of tissue or myelin loss. Note that most of the damage affects the gracile fasciculi and the dorsal horn, whereas the ventral funiculus and horn have less damage (A). The cystic lesions and tissue damage were extended at 6 weeks after SCI and cyst sizes were greater in the control-injured group than the Oxycyte-injured group. Cord sections of Oxycyte-injured animals have reduced lesion size (B) and better myelin and Nissl preservation (D), whereas SCI-control and SCI-saline groups have larger cysts (B) and greater myelin loss and damage Nissl (B), whereas in the section of animals that sustained laminectomy, no lesions or cavities were detectable and no myelin or Nissl loss (B and D). Note that myelin sheath collapsed into the area formally occupied by the axon in the SCI-control and saline groups, whereas it was significantly less in the Oxycyte group. Representative transverse spinal cord sections after T10 contusion injury were taken at 1 mm caudal to the lesion epicenter. Photomicrographs sections taken at 2.5× magnification (A and B). Arrow heads in (B) indicated the enlarged area magnified 40× in (D). (E) Graph depicting area of white matter in spinal cord sections caudal to lesion epicenter of Oxycyte- and saline-treated animals as well the SCI group that underwent spinal cord contusion at T9. Data represent means±standard error (n=3). Oxycyte-treated animals 42 days postoperatively had significantly more spared white matter than saline-treated animals; there was a statistically significant difference between the saline and Oxycyte groups (p=0.022, Welch's two-tailed t-test comparison). Representative micrograph of spinal cord cross-sections 42 days after contusion is presented for each group above their statistical value. SCI, spinal cord injury; POD, postoperative day. Color image is available online at www.liebertpub.com/neu

FIG. 3.

Oxycyte reduces apoptotic cell death. Representative photomicrographs (20×) of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining illustrating apoptotic cell death after SCI. Spinal cords from rats after 7 days postoperatively were processed and stained as described in the Methods section. Oxycyte administration suppressed SCI-induced apoptosis rostral (D) and caudal from the lesion epicenter (H). The numbers of TUNEL-positive cells (arrowheads) are significantly higher in SCI (B and F) and saline-injured groups (C and G). Oxcycyte treatment protected spinal cord cells in both rostral (D) and caudal sections (H) from the lesion epicenter. Sections analyzed were from 2 mm rostral (A–D) and caudal to the lesion epicenter (E–H). Laminectomy control showed only occasional labeled cells in the spinal cord sections (A and E). SCI, spinal cord injury.

FIG. 4.

Active caspase 3 colocalize with apoptotic cells. High-power photomicrograph (1000×) of spinal cord sections that were TUNEL stained, showing characteristic apoptotic cell death (chromatin condensation and DNA fragmentation) in neuron (*) and oligodendrocytes indicated by arrowhead (A). Coimmunofluorescence of TUNEL with cleaved caspase 3 confirmed that TUNEL-reactive cells were indeed apoptotic (B). Time course of apoptotic cell death after SCI presented in bar graph showing the number of TUNEL-positive nuclei 1–42 days postinjury (C) Oxcycyte-treated animals presented few apoptotic cell deaths, compared to control- and saline-injured animals (n=3 cords in each group). Bar graph presenting the number of TUNEL-positive cells in spinal cord sections 7 days postinjury. Apoptotic cells were calculated for rostral and caudal 1 mm to the lesion epicenter (D). Note that administration of Oxycyte significantly reduced apoptotic cell number in both rostral and caudal sections, compared to the saline group. Asterisks indicate that means are significantly different from the saline-control group at the specified times after SCI (D). TUNEL, deoxynucleotidyl transferase enzyme TdT nick end labeling; SCI, spinal cord injury.

FIG. 5.

Oxcycyte and saline treatment results in a trend toward motor-enhanced function. Time course of functional recovery after spinal cord injury (SCI). (A) Graphical data represent mean open-field BBB scores of the vehicle-saline (n=5) and oxycyte-treated animals (n=5) and contused animals (n=5) during the 6-week experimental period. All groups displayed the same partial recovery (BBB score=10) during the first 14 days, then diverged over the last 4 weeks, with Oxcycyte and saline treatment resulting in a trend toward enhanced function. (B) Locomotor function was also tested by the inclined plane test on the same groups of animals as assessed in (A). In most cases, motor function recovered more in the Oxycyte- and saline-treated groups than in the injured control groups. The Oxycyte- and saline-treated groups showed significant behavioral improvement (A) and significant ability to climb a steeper inclined board, compared to injured untreated animals (B). BBB, Basso-Beattie-Bresnahan scale.