Protective Effects Of Nicotinamide Adenine Dinucleotide And XIAP Gene On Ototoxicity Induced By Manganese In Cochlear Organotypic Cultures Of Neonatal Rat

Objectives:(1) To establish the animal model of manganese (Mn) ototoxicity and observe the toxic effect of manganese chloride (MnCl2) on rat cochlea in vitro as well as its does-response relationship.(2) To evaluate the effectiveness of nicotinamide adenine dinucleotide (NAD) in treatment of Mn induced ototoxicity and to observe apoptosis of cochlear cells induced by MnCl2and performance of NAD in anti-apoptosis process.(3) To evaluate the safety and effectiveness of an improved hydroxyapatite nanoparticle vector mediated delivery of the XIAP gene (X-linked inhibitor of apoptosis protein, a member of inhibitor of apoptosis protein family) in treatment of Mn induced ototoxicity.Methods:(1) Postnatal day3SASCO Sprague-Dawley rats were used for this study. The cochleae were carefully dissected out and cultured overnight. On the second day, the cultures were treated with or without MnCl2at concentration from0.5mM to3.0mM for48h. Specimens were immune stained and examined under the laser confocal inverted fluorescence microscope, and cochleograms were prepared from cochlea cultures.(2) After dissection and overnight culture, the cultures were exposed to MnCl2at concentrations from0.5mM to3.0mM in the presence or absence of20mM NAD for48h; and the blank control group was treated without MnCl2or NAD. Specimens were immune stained and examined under the laser confocal inverted fluorescence microscope, and cochleograms were prepared from cochlea cultures. The number of surviving auditory nerve fiber (ANF) per unit length of cochlear basilar membrane and spiral ganglion neuron (SGN) per unit volume of cochlear ganglion in each group was calculated and performed with statistical tests.(3) After dissection and overnight culture, the cultures were exposed to MnCl2in the presence or absence of20mM NAD; and the blank control group was treated without MnCl2or NAD.6hours following treatment, the unfixed specimens were labeled with caspase; and9hours after treatment, TUNEL staining was performed. Specimens were then examined under the laser confocal inverted fluorescence microscope, and the ratio of caspase or TUNEL labeled SGN in each group was calculated and performed with statistical tests.(4) Improved nano-hydroxyapatite (PEG-PEI-nHAT) were synthesized by chemical coprecipitation-hydrothermal synthesis method and combined to the therapeutic gene XIAP to prepare the nano gene-vector compound PEG-PEI-nHAT-pEGFPNl-XIAP. In safety assessment, cultures were treated in the present or absence of500μl PEG-PEI-nHAT-pEGFPN1-XIAP at concentration of1μg/μl for48h; in effectiveness evaluation, cultures were exposed to MnCl2at concentrations from0.5mM to3.0mM in the presence or absence of500μl PEG-PEI-nHAT-pEGFPN1-XIAP at concentration of1μg/ul for48h. Specimens were immune stained and examined under confocal microscope, and the number of surviving SGN in each group was calculated and performed with statistical tests.Results:(1) In controlled group, outer hair cell (OHC) and inner hair cell (IHC) were arranged in orderly and there was no hair cell loss or damage; ANF of SGN radiating outward towards the hair cell (HC) were organized into smooth, thick fascicles; and normal SGN have large and oval-shaped soma. In treatment group, the density of HC, ANF and SGN were decreasing as the concentrations of Mn increased; the actin in the cuticular plate and the stereocilia of HC were reduced, the ANF started to appear thin and fragment, and SGN soma were shrunken and fragmented, illustrating characteristics of cell apoptosis. Cochleograms showed that the ratio of HC loss was increasing from20%to70%approximately.(2) Compared with the control (cochlear cultures treated with0.5-3.0mM MnCl2for48h), NAD clearly prevented the toxic actions of Mn on cochlear culture in treatment group (treated with0.5-3.0mM MnCl2combined with20mM NAD for48h). The addition of20mM NAD to the Mn treated cultures resulted in a substantial increase in ANF survival along with reduced blebbing and fragmentation of the fibers. Soma and nucleolus shrinkage and SGN losses were less severe than in cultures treated with a similar concentration on Mn alone. Under each Mn concentration, the survival rate of SGN and ANF in treatment group was significantly greater than in the control group (p<0.05).(3) TUNEL staining was minimal in control cultures but was frequently observed in SGN cultured for9h in1.0mM Mn. Adding20mM NAD to cultures treated with1.0mM Mn reduced the number of TUNEL positive SGN. Caspase-3, caspase-8and casepase-9labeling was seldom observed in SGN in control samples; in contrast, caspase labeling was seen in many SGN in cultures treated with1.0mM Mn for6h; caspase labeling occurred predominantly in SGN with shrunken and/or distorted soma; with1.0mM Mn plus20mM NAD, there was a noticeable reduction in caspase-3, caspase-8and caspase-9labeling compared to1.0mM Mn alone. The ratios of TUNEL and caspase positive SGN in treatment group were both significantly less than in the control group (p<0.05).(4) In security assessment of PEG-PEI-nHAT-pEGFPN1-XIAP, SGN have large and oval-shaped soma in both group, and there was no significant SGN loss or damage in culture treated with PEG-PEI-nHAT-pEGFPN1-XIAP for48h. XIAP was expressed in SGN and peripheral cells, presenting green fluorescence. There was no statistical difference between the number of survived SGN in control and treatment group (p>0.05). In effectiveness evaluation, SGN soma were shrunken and fragmented and cell loss was servere in control group as MnCl2concentration increased from0.5to3.0mM. In contrast, karyopyknosis, soma fragment and cell loss still existed but was less severe in culture treated with0.5-3.0mM MnCl2plus500μl PEG-PEI-nHAT-pEGFPN1-XIAP compared with the control, and XIAP was expressed in SGN and labeled. Under each concentration of Mn, the survival rate of SGN in culture treated with PEG-PEI-nHAT-pEGFPN1-XIAP and Mn was significantly greater than in the control group (p<0.05), indicating that XIAP gene may protect cochlear culture from Mn-induced ototoxicity.Conclusions:(1) Mn causes significant toxicity on HC, ANF and SGN in SD rat in does-dependent manner. The extent of pathological damage exacerbates as the concentration of Mn increases.(2) NAD can prevent toxicity of Mn on ANF and SGN of rat cochlea. In lower concentration, NAD can prevent toxicity of Mn on HC of rat cochlea.(3) The pathophysiology of Mn toxicity on cochlea is due in part to the upregulation of caspase-3,-8and-9, DNA fragmentation and induction of apoptosis. NAD suppresses Mn-induced upregulation of caspase, DNA fragmentation and apoptosis.(4) The improved nano-hydroxyapatite vector mediated delivery of the XIAP gene (PEG-PEI-nHAT-pEGFPN1-XIAP) demonstrates no apparent acute toxic effect on SGN and XIAP gene can potentially prevents toxicity of Mn on SGN.