This study aimed to modify the mixed and purified culture of rat retinal ganglion cells (RGCs) in vitro. The retinae of 1-3 day old Sprague-Dawley (SD) rats were separated bluntly into two layers: inner layer and outer layer, under a surgical microscope. Retinal cells isolated from different layers (inner layer, outer layer and whole retinal tissue) by using enzyme dissociation method were cultured in F12/DMEM medium containing 15% FBS. After 3-day culture, the RGCs in the retinal cells obtained from mixed culture of inner, outer, and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1, and the rate of RGCs to retinal cells (RGCs%) was calculated. Two monoclonal antibodies, anti-macrophages/granulocytes (OX-41) against rat macrophage and antibody against rat Thy-1.1 (OX-7), were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure (purification in situ). Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15% FBS. Immunocytochemical staining for Thy-1.1, MTT, and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs. The results showed: (1) Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs% was (19.9±1.2)%, (0.5±0.2)%, and (6.2±1.7)% respectively in the mixed culture of inner, outer, and whole retinal tissue, with differences being significant (P<0.05); (2) fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs%, RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method; (3) the viability of purified RGCs seeded at high density was increased and the cells developed complex intercellular networks. The viability of RGCs was declined with the decreasing seeding density, and most cells presented round or oval in shape with thin neurites. It was concluded that: (1) RGCs% in the inner layer retina was higher than that in the outer layer retina; (2) RGCs obtained by in situ purification had more neurite outgrowth and lower mortality than those by conventional two-stepped immunopanning procedure; (3) the viability of purified RGCs could be increased by increasing cell seeding density to some extent.

Haemophagocytic lymphohistiocytosis (HLH) is a clinico-pathologic entity caused by increased proliferation and activation of benign macrophages with haemophagocytosis throughout the reticulo-endothelial system. Virus-associated HLH is a well-recognised entity. Although majority of parvovirus B19 associated HLH does not require any specific treatment and carries good prognosis, outcome of children is worse than adults. We report here a case of HLH associated with acute parvovirus B19 infection in a young healthy patient with underlying hereditary spherocytosis, with bone marrow findings typical of parvovirus infection. Although this patient had spontaneous recovery of cell counts, he succumbed due to complication from prolonged ventilation. Unexpectedly, his immunoglobulin levels were inappropriately normal despite on-going ventilator associated pneumonia, which reflects inadequate humoral immune response towards infection.
Parvovirus

This study aimed to modify the mixed and purified culture of rat retinal ganglion cells (RGCs) in vitro.The retinae of 1-3 day old Sprague-Dawley (SD) rats were separated bluntly into two layers:inner layer and outer layer,under a surgical microscope.Retinal cells isolated from different layers (inner layer,outer layer and whole retinal tissue) by using enzyme dissociation method were cultured in F12/DMEM medium containing 15％ FBS.After 3-day culture,the RGCs in the retinal cells obtained from mixed culture of inner,outer,and whole retinal tissue were identified by immunocytochemical staining of Thy-1.1,and the rate of RGCs to retinal cells (RGCs％) was calculated.Two monoclonal antibodies,anti-macrophages/granulocytes (OX-41) against rat macrophage and antibody against rat Thy-1.1 (OX-7),were used to purify RGCs by either a conventional or modified two-stepped immunopanning procedure (purification in situ).Purified RGCs were seeded at different cell density and cultured in F12/DMEM medium containing 15％ FBS.Immunocytochemical staining for Thy-1.1,MTT,and PI-Hoechst33342 fluorescence imaging were used to identify the purity and the viability of RGCs in purified culture of RGCs.The results showed:(1) Immunocytochemistry of different retinal tissue layers culture revealed that the RGCs％ was (19.9±1.2)％,(0.5±0.2)％,and (6.2±1.7)％ respectively in the mixed culture of inner,outer,and whole retinal tissue,with differences being significant (P＜0.05); (2)fluorescent double staining of Hoechst33342 and PI indicated that with the same RGCs％,RGCs obtained from purification in situ grew well with more neurite outgrowth than those by the conventional two-stepped immunopanning method; (3) the viability of purified RGCs seeded at high density was increased and the cells developed complex intercellular networks.The viability of RGCs was declined with the decreasing seeding density,and most cells presented round or oval in shape with thin neurites.It was concluded that:(1) RGCs％ in the inner layer retina was higher than that in the outer layer retina;(2) RGCs obtained by in situ purification had more neurite outgrowth and lower mortality than those by conventional two-stepped immunopanning procedure; (3) the viability of purified RGCs could be increased by increasing cell seeding density to some extent.