The Effects And Mechanisms Of Histone Deacetylase Inhibitor DMA-PB On Traumatic Brain Injury In Rats

Objective:Traumatic brain injury(TBI) produces a number of pathologies including inflammation.The inflammatory cascade is characterized by activation of microglia and proinflammatory cytokines which can exacerbate other pathologies.Pharmacological therapy that can modulate pro-inflammatory cytokines or microglia activation could have potential efficacy in TBI.A number of studies have focused on histone deacetylase(HDAC) inhibitors as novel therapeutics in a variety of central nervous system(CNS) diseases and showed anti-inflammatory effects.However no data have been reported on TBI.A recent study in experimental pediatric TBI reported a decrease in hippocampal CA3 histone H3 acetylation after injury.These changes were attributed to documented upstream excitotoxic and stress cascades associated with TBI.HDAC inhibitors may contribute to prevention and treatment of TBI.The aim of this study was to evaluate the possible protective effects of DMA-PB,a novel HDAC inhibitor,on TBI.Methods:Drug was administered intraperitoneally(i.p.) immediately after TBI.Coronal brain sections were stained with acetyl-histone H3 antibody,OX-42 antibody or Fluoro-Jade B.Measurement of proinflammatory cytokines(TNFα and IL-1β),acquisition function and delayed neuronal degeneration was assessed with Enzyme-Linked ImmunoSorbent Assay(ELISA),Morris Water Maze(MWM) and cresyl violet staining techniques.The region of interest (ROI) was focused on hippocampus.Results:Histone acetylation was assessed by Acetyl-Histone H3 immunohistochemistry and measured by relative optical density(OD).The result showed that lower ROD was observed in the hippocampus CA2/3 region ipsilateral to FPI compared to sham group.The ROD among DMA-PB dosage treatment groups was not significantly different from the sham group.Microglia changes was carried out by using OX-42 immunohistochemistry and assessed by quantification of four classifications of microglia to identify inflammatory response.The sham group had a preponderance of resting microglia density that was significantly greater than any of the injury groups.No activated or phagocytic microglia was detected in the sham group.There were no significant differences in mean density of activated microglia between the injury groups.The density of phagocytic microglia was significantly different between injury groups.The DMA-PB treatments had significantly lower density of phagocytic microglia compared to the vehicle-treated TBI group.Neuronal degeneration was analyzed by using Fluoro-Jade B histofluorescence.The results showed that there was a trend for the DMA-PB treatment to reduce the number of ipsilateral degeneration neurons compared to vehicle with the highest dose(25 mg/kg) producing the largest reduction in neurodegeneration.Detection of TNFαand IL-1βwas made using enzyme-linked immunosorbent assay(ELISA).In vehicle-treated injured rats,the ipsilateral hemisphere showed a significant increase in TNFαand IL-1βlevels.The profound increase in pro-inflammatory cytokines associated with trauma was significantly reduced by DMA-PB treatment compared with levels in vehicle-treated injured rats.We further investigated more chronic outcome after TBI.Acquisition of a spatial memory was measured by using Morris water maze.The vehicle-treated TBI group showed a significant increase in the latencies(time to find a hidden platform) across testing days compared to sham control.However,DMA-PB treated group(25mg/kg) demonstrated significant improvement in decreased latencies. Delayed neuronal injury was analyzed by using Cresyl violet staining.In the sham group the neurons were normal,their borders were distinct and cell membrane integrity was preserved.Nucleus borders were regular and nucleolus was distinctively observed.In the injury groups,marked shrinkage was observed in the pyramidal neurons of CA2/3 region and increased irregularity was determined in cell and nucleus borders.Typical multiple row cell arrangements were disturbed and the number of cells was decreased.The effect of DMA-PB treatment on neuron density was examined by quantitative histopathological analysis.In vehicle group,hippocampal neuron density was significantly decreased in comparison with the sham group.However,treatment with DMA-PB (25mg/kg) significantly preserved the neurons of hippocampal regions as compared to vehicle group.Conclusions:the present study provided further evidence for the deacetylation of histone H3 by TBI and demonstrated that the novel HDAC6 inhibitor,DMA-PB,reversed the deacetylation measured in the hippocampus likely through an indirect action and robustly attenuated the inflammatory microglia response and production of inflammatory cytokines,produced a trend toward reduced neuronal degeneration in the hippocampus at early time after injury.DMA-PB significantly exhibited more chronic protective outcome characterized by efficiently decreasing delayed neuronal injury and improving spatial memory function.Our findings may provide a primary evidence for the possible benefits of HDAC inhibitors in TBI.We also provide some basic materials for novel HDAC inhibitor,DMA-PB.Further studies of HDAC inhibitors appear warranted for clarification of the underlying mechanisms of action for potential development of therapeutics for patients suffering from TBI.