NNZ-2566 treatment inhibits neuroinflammation and pro-inflammatory cytokine expression induced by experimental penetrating ballistic-like brain injury in rats

Abstract

Background: Inflammatory cytokines play a crucial role in the pathophysiology of traumatic brain injury (TBI), exerting either deleterious effects on the progression of tissue damage or beneficial roles during recovery and repair. NNZ-2566, a synthetic analogue of the neuroprotective tripeptide Glypromate, has been shown to be neuroprotective in animal models of brain injury. The goal of this study was to determine the effects of NNZ-2566 on inflammatory cytokine expression and neuroinflammation induced by penetrating ballistic-like brain injury (PBBI) in rats.

Methods: NNZ-2566 or vehicle (saline) was administered intravenously as a bolus injection (10 mg/kg) at 30 min post-injury, immediately followed by a continuous infusion of NNZ-2566 (3 mg/kg/h), or equal volume of vehicle, for various durations. Inflammatory cytokine gene expression from the brain tissue of rats exposed to PBBI was evaluated using microarray, quantitative real time PCR (QRT-PCR), and enzyme-linked immunosorbent assay (ELISA) array. Histopathology of the injured brains was examined using hematoxylin and eosin (H&E) and immunocytochemistry of inflammatory cytokine IL-1beta.

Results: NNZ-2566 treatment significantly reduced injury-mediated up-regulation of IL-1beta, TNF-alpha, E-selectin and IL-6 mRNA during the acute injury phase. ELISA cytokine array showed that NZ-2566 treatment significantly reduced levels of the pro-inflammatory cytokines IL-1beta, TNF-alpha and IFN-gamma in the injured brain, but did not affect anti-inflammatory cytokine IL-6 levels.

Conclusion: Collectively, these results suggest that the neuroprotective effects of NNZ-2566 may, in part, be functionally attributed to the compound's ability to modulate expression of multiple neuroinflammatory mediators in the injured brain.

Figure 1

NNZ-2566 treatment decreased inflammatory leukocyte filtration (H & E staining) after PBBI. Neutrophils (black arrows) and large macrophage-like cells (white arrows) were prominent in area surrounding the lesion cavity 3 days following PBBI (A, C). NNZ-2566 treatment (B, D) inhibited the infiltration of neutrophils and macrophage-like cells (B and D). By day 7, density of cellular infiltrate surrounding the lesion cavity was also reduced with the NNZ-2566 treatment (F) compared to the vehicle treatment (E).

Figure 2

Representative microarray images of inflammatory cytokine gene arrays from sham-PBBI, PBBI, and NNZ-2566 treated PBBI rats 4 hours after the injury are shown. GAPDH and RPL32 served as internal controls. Genes for which robust changes in mRNA expression were observed are indicated by the arrows.

Figure 3

Time-course of gene expression as measured by qRT-PCR. The mRNA levels are given as the mean ± standard error (n = 6 per group) normalized to RPL32 levels for each sample. *P < 0.05 as compared to the sham and # P < 0.05 as compared to PBBI treated with the vehicle.

Figure 4

Multiplex cytokine array analysis of cytokine levels following PBBI. NNZ-2566 treatment is similar to that listed in legend of figure 3. (A) Schematic indicating the orientation of cytokines (IL-1β, IL6, INF-γ, and TNF-α) and representative images of chemiluminiscent intensity from the multiplex array (4 hours after PBBI). (B) Quantitative analysis from the multiplex array measurements. The cytokine levels are given as the mean ± standard error (n = 6 per group). *P < 0.05 as compared to the sham, and # P < 0.05 as compared to PBBI with the vehicle.

Figure 5

PBBI-induced IL-1β immunoreactivity in vehicle (A) or NNZ-2566 (B) treated rats at 3 (C and D) or 7 days (E and F). IL-1β positive cells are visible in the region surrounding the lesion cavity at 3 days following PBBI (C) which are reduced following NNZ-2566 treatment (D). Panel E and F are representative photomicrographs of injured hemisphere at 7 days for PBBI (E) or PBBI + NNZ-2566 (F).