Treatment Of Epilepsy: The GABA-transaminase Inhibitor, Vigabatrin, Induces Neuronal Plasticity In The Mouse Retina And Suppressed By Darkness

Epilepsy affects 1%of the world population and can be life threatening. Vigabatrin,(gamma-vinyl GABA,or VGB),was widely prescribed for the treatment of epilepsy because it reduces the occurrence of seizures by increasing the GABA concentration,the main inhibitory neurotransmitter of the central nervous system.Vigabatrin irreversibly inhibits GABA transaminase,the GABA degrading enzyme.Unfortunately,chronic administration of VGB was found to induce a bilateral constriction of the visual field in 10 to 40%of treated patients.There have been several reports of altered visual acuity,colour discrimination or contrast sensitivity whereas other studies,such as the longitudinal cohort studies sponsored by the National Health Service of Scotland,have demonstrated preservation of central visual acuity and colour vision in patients with bilateral constriction.Retinal damages were described in albino rats and they included cone photoreceptor damage,disorganization of the photoreceptor layer and gliosis indicated by increased GFAP immunocytochemical labelling.The pathological changes in albino rats did not fully explain human symptoms.To further investigate the vigabatrin-elicited retinal toxicity and take advantage of genetically modified animals,we have examined retinal changes in vigabatrin-treated albino mice.Although the photoreceptor layer was occasionally disorganized,this symptom was less often encountered than in vigabatrin-treated rats.The cone photoreceptor damage could be detected as it expressed in albino rats,and retinal glial cells were not more intensely labelled in vigabatrin-treated mice than in control albino animals.However,rod photoreceptors were found to withdraw their synapses from the outer plexiform layer to their cell body as indicated by bassoon immunostaining of their synaptic ribbons or VGLUT1-immunolabelling of synaptic vesicles.Cone photoreceptors maintained their terminals in the outer plexiform layer.In parallel,not only rod ON bipolar cells and horizontal cells but also cone ON bipolar cells exhibited dendrites sprouting into the photoreceptor nuclear layer. Contacts between withdrawing rod synaptic terminals and the growing postsynaptic neurones were not always maintained.This plasticity of retinal neurones which could explain the constriction of the visual field appeared as the main retinal damage in vigabatrin-treated albino mice.VGB-elicited photoreceptor degeneration was reported to be light-dependent in vitro.This is consistent with the selective VGB sensitivity of albino but not pigmented animals.Base on these findings, we kept a group of albino mice in darkness during chronic VGB treatment to further assess the light dependence of VGB-elicited toxicity.As we hypothesized,the plasticity of retinal neurons was completely suppressed by darkness.Purpose:(1)To investigate the vigabatrin toxicity on albino mouse retina, evaluate the manifestation of retinal toxicity in diverse genetical modified animals.(2)To detect the correlation between phototoxicity and vigabation-elicited retinal damage,explore the potential management of vigabatrin toxicity on retina.Methods:Part 1.Twenty six BALB/c male mice were separated to two groups. Sixteen of them received daily intraperitoneal injection of vigabatrin and lasted for thirty days.VGB was dissolved in 0.9%NaCl at a concentration of 100mg/ml and final injection dose was 250mg/kg as described for animal treatment of epilepsy.Ten animals received 0.1ml 0.9%NaCl daily injection as control group.On day 31,animals were sacrificed and enucleated,after removing the cornea and lens,eye cups were fixed and cryosection were prepared in 8-10μm thickness.The sections were immunostained separately with Anti-mouse cone arrestin (1:20,000,Luminaire junior,LUMIj),Anti-protein kinase C alpha(PKCα)(1:2000,Sigma),Anti-VGlur1,(1:2000,Chemicon),Anti-GoαK-20(1:100,Santa Cruz),Anti-calbindin D-28K(1:500,Chemicon),Anti-Goα(1:2000,Chemicon),Anti-bassoon(1:100,StrseeGen),DAPI(1:500, Molecular Probes,Eugene,OR),the second antibody is Goat anti-rabbit IgG or rabbit anti-mouse IgG conjugated to either Alexa TM594 or Alexa TM488 (Molecular Probes,Eugene,OR)。Final results were taken by Ropper scientific camera(Photometrics cool SNAP TM FX).Part 2.Thirty BALB/c male mice were separated to three groups.Ten animals received VGB daily intraperitoneal injection were kept in normal day-night cycle situation as VGB treatment group;another ten mice were kept in darkness during the whole process of VGB intraperitoneal injection as VGB darkness group;the residual ten animals which accept vehicle solution injection were also kept in normal day-night cycle situation as control group.The whole period of experiment also last thirty days,VGB concentration and dose of injection were the same as Part 1.On day 31, animals were sacrificed and enucleated,steps of cryosections and inmunostaining were repeated as Part 1.Final results were also recorded with Ropper scientific camera.Results:Part 1.(1) Areas with a highly disorganized outer nuclear layer were observed in vigabatrin-treated albino mice but not in all animals(5 of 16 treated mice)as treated rats.(2) Staining of cone arrestin showed outer segments of cones were altered in restricted areas as in VGB-treated rats but their terminals remained intensely labeled in out plexiform layer.(3) GFAP staining was not increased by the vigabatrin treatment.(4) Rod terminals were withdraw into the ONL close to photoreceptor nuclei in all vjgabatrin treated mice.(5)Bipolar cells sprouted their dendrites form out plexiform layer to the out nuclear layer in all VGB treated animals.(6) In all VGB-treated animals,horizontal extended their dendrites from out plexiform layer into the out nuclear layer like bipolar cells.(7)Double labelling indicated that the rod terminal withdrawal was associated with a loss of synaptic transmission between photoreceptors and their postsynaptic neurons.(8) Double labelling indicated that the connections between cone terminals and bipolar dendrites were lost with bipolar sprouting and cone terminals stable in OPL.Part 2.(1) In VGB darkness group,withdraw of photoreceptor terminal were completely suppressed.(2) VGB-elicited sprouting of bipolar cell dendrites disappeared in dark situation.(3) The horizontal dendrites migration were also inhibited in VGB darkness group.Conclusions:(1) Vigabatrin treatment in albino mice could represented by the retinal architecture disorganization and partial out segment loss in some degree,which is similar to the rat retina damage.(2) GFAP staining is the prominent mark in rat retina damage,on contrast,this staining is unspecific reaction both in VGB treated mice and control group which means the glial staining is not an indicator of mouse retina impairment.(3) The hallmark of vigabatrin hazard on mouse retina is the retinal neuro-plasticity:rod dendrites withdraw and bipolar axons sprouting accompany with horizontal cell terminals.(4) Darkness could eliminate the distinctive neuro-plasticity completely on albino mouse retina demonstrated vigabatrin-induced damage is correlate with phototoxicity, which indicated a potential way of prevention of vigabatrin injury in clinic.