BMPRⅡ^(+)neural precursor cells isolated and characterized from organotypic neurospheres:an in vitro model of human fetal spinal cord development

Roof plate secretion of bone morphogenetic proteins(BMPs)directs the cellular fate of sensory neurons during spinal cord development,including the formation of the ascending sensory columns,though their biology is not well understood.Type-ⅡBMP receptor(BMPRⅡ),the cognate receptor,is expressed by neural precursor cells during embryogenesis;however,an in vitro method of enriching BMPRⅡ^(+)human neural precursor cells(hNPCs)from the fetal spinal cord is absent.Immunofluorescence was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRⅡand leukemia inhibitory factor(LIF).Regions of highest BMPRⅡ^(+)immunofluorescence localized to sensory columns.Parenchymal and meningeal-associated BMPRⅡ^(+)vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers,CD34/CD39.LIF immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons,mirroring the expression of LIF receptor/CD118.A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages,while synergistically increasing the proportion of neurospheres with a stratified,cytoarchitecture.These neurospheres were characterized by BMPRⅡ^(+)/MAP2ab^(+/–)/βⅢ-tubulin^(+)/nestin^(–)/vimentin^(–)/GFAP^(–)/NeuN^(–)surface hNPCs surrounding a heterogeneous core ofβⅢ-tubulin^(+)/nestin^(+)/vimentin^(+)/GFAP^(+)/MAP2ab^(–)/NeuN^(–)multipotent precursors.Dissociated cultures from tripotential neurospheres contained neuronal(βⅢ-tubulin^(+)),astrocytic(GFAP+),and oligodendrocytic(O4+)lineage cells.Fluorescence-activated cell sorting-sorted BMPRⅡ^(+)hNPCs were MAP2ab^(+/–)/βⅢ-tubulin^(+)/GFAP^(–)/O4^(–)in culture.This is the first isolation of BMPRⅡ^(+)hNPCs identified and characterized in human fetal spinal cords.Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres,characterized by surface BMPRⅡ^(+)hNPCs.Our study has provided a new methodology for an in vitro model capable of amplifying human fetal spinal cord cell numbers for>10 passages.Investigations of the role BMPRⅡplays in spinal cord development have primarily relied upon mouse and rat models,with interpolations to human development being derived through inference.Because of significant species differences between murine biology and human,including anatomical dissimilarities in central nervous system(CNS)structure,the findings made in murine models cannot be presumed to apply to human spinal cord development.For these reasons,our human in vitro model offers a novel tool to better understand neurodevelopmental pathways,including BMP signaling,as well as spinal cord injury research and testing drug therapies.

Postnatal development of rat retina:a continuous observation and comparison between the organotypic retinal explant model and in vivo development

The organotypic retinal explant culture has been established for more than a decade and offers a range of unique advantages compared with in vivo experiments and cell cultures.However,the lack of systematic and continuous comparison between in vivo retinal development and the organotypic retinal explant culture makes this model controversial in postnatal retinal development studies.Thus,we aimed to verify the feasibility of using this model for postnatal retinal development studies by comparing it with the in vivo retina.In this study,we showed that postnatal retinal explants undergo normal development,and exhibit a consistent structure and timeline with retinas in vivo.Initially,we used SOX2 and PAX6 immunostaining to identify retinal progenitor cells.We then examined cell proliferation and migration by immunostaining with Ki-67 and doublecortin,respectively.Ki-67-and doublecortin-positive cells decreased in both in vivo and explants during postnatal retinogenesis,and exhibited a high degree of similarity in abundance and distribution between groups.Additionally,we used Ceh-10 homeodomain-containing homolog,glutamate-ammonia ligase(glutamine synthetase),neuronal nuclei,and ionized calcium-binding adapter molecule 1 immunostaining to examine the emergence of bipolar cells,Müller glia,mature neurons,and microglia,respectively.The timing and spatial patterns of the emergence of these cell types were remarkably consistent between in vivo and explant retinas.Our study showed that the organotypic retinal explant culture model had a high degree of consistency with the progression of in vivo early postnatal retina development.The findings confirm the accuracy and credibility of this model and support its use for long-term,systematic,and continuous observation.

Engineering vascularized organotypic tissues via module assembly

Adequate vascularization is a critical determinant for the successful construction and clinical implementation of complex organotypic tissue models. Currently, low cell and vessel density and insufficient vascular maturation make vascularized organotypic tissue construction difficult,greatly limiting its use in tissue engineering and regenerative medicine. To address these limitations, recent studies have adopted pre-vascularized microtissue assembly for the rapid generation of functional tissue analogs with dense vascular networks and high cell density. In this article, we summarize the development of module assembly-based vascularized organotypic tissue construction and its application in tissue repair and regeneration, organ-scale tissue biomanufacturing, as well as advanced tissue modeling.

Lipopolysaccharide preconditioning induces protection against lipopolysac-charide -induced neurotoxicity in organotypic midbrain slice culture

Objective To identify the protective effect of lipopolysaccharide （LPS） preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms. Methods After cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations （0, 1, 3, 6 or 10 ng/mL） of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was detelmined by measurement of the activity of lactic acid dehydrogenase （LDH）. Tyrosine hydroxylase-immunoreactive （TH-IR） neurons and CD 1 1 b/c equivalent-immunoreactive （OX-42-IR） microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-α （TNF-α levels in the culture media were detected by enzymelinked immunosorbent assays （ELISA）. Results In the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191± 12 in the control slices to 46±4, and the LDH activity elevated obviously （P 〈 0.01）, along with remarkably increased number of OX-42-IR cells and production of TNF-α （P 〈 0.01）. Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss （the number of TH-IR neurons increased to 126± 12 and 180± 13, respectively） and markedly reduced LDH levels （P 〈 0.05）, accompanied by significant decreases of OX-42-IR microglia activation and TNF-α production （P 〈 0.05）. Conclusion Low-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-α production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson＇s disease.

OTOTOXIC EFFECTS OF CARBOPLATIN IN ORGANOTYPIC CULTURES IN CHINCHILLAS AND RATS

Carboplatin, a second-generation platinum chemotherapeutic drug, is considerably less ototoxic than cisplatin. While common laboratory species such as mice, guinea pigs and rats are highly resistant to carboplatin ototoxicity, the chinchilla stands out as highly susceptible. Moreover, carboplatin causes an unusual gradient of cell death in chinchillas. Moderate doses selectively damage type I spiral ganglion neurons (SGN) and inner hair cells (IHC) and the lesion tends to be relatively uniform along the length of the cochlea. Higher doses eventually damage outer hair cells (OHC), but the lesion follows the traditional gradient in which damage is more severe in the base than the apex. While carboplatin ototoxicity has been well documented in adult animals in vivo, little is known about its in vitro toxicity. To elucidate the ototoxic effects of carboplatin in vitro, we prepared cochlear and vestibular organotypic cultures from postnatal day 3 rats and adult chinchillas. Chinchilla cochlear and vestibular cultures were treated with carboplatin concentrations ranging from 50 μM to 10 mM for 48 h. Consistent with in vivo data, carboplatin selectively damaged IHC at low concentrations (50-100 μM). Surprisingly, IHC loss decreased at higher doses and IHC were intact at doses exceeding 500 μM. The mechanisms underlying this nonlinear response are unclear but could be related to a decrease in carboplatin uptake via active transport mechanisms (e.g., copper). Unlike the cochlea, the carboplatin dose-response function increased with dose with the highest dose destroying all chinchilla vestibular hair cells. Cochlear hair cells and auditory nerve fibers in rat cochlear organotypic cultures were unaffected by carboplatin concentrations <10 μM; however, the damage in OHC were more severe than IHC once the dose reached 100 μM. A dose at 500 μM destroyed all the cochlear hair cells, but hair cell loss decreased at high concentrations and nearly all the cochlear hair cells were present at the highest dose, 5 mM. Unlike the nonlinear dose-response seen with cochlear hair cells, rat auditory nerve fiber and spiral ganglion losses increased with doses above 50 μM with the highest dose destroying virtually all SGN. The remarkable species differences seen in vitro suggest that chinchilla IHC and type I SGN posse some unique biological mechanism that makes them especially vulnerable to carboplatin toxicity.

Lead neurotoxicity in rat cochlear organotypic cultures

Lead is a major environmental toxicant throughout the world.Lead can induce severe neurotoxicity including irreversible hearing impairment.Many in vivo studies have shown that lead damages the auditory nervous system,but has little or no effect on cochlear sensory hair cells.To gain insights on lead ototoxic and neurotoxic effects in vitro,lead acetate (LA) was applied to postnatal day 3-4 rat cochlear organotypic cultures for 24 or 72 h with doses of 0.1,0.5,1,2 or 4 mM.After 24 or 72 h treatment with lead acetate,nearly all of cochlear sensory hair cells were intact.However,after 72 h treatment,the peripheral auditory nerve fibers projecting to the hair cells and the spiral ganglion neurons (SGN) were damaged when lead concentration exceeded 2 mM.Our results indicated that 72 h treatment with only the high doses (> 2 mM) of lead actate damaged SGNs and peripheral nerve fibers;hair cells remained structurally intact even after 4 mM treatment.These results show that lead primarily damages cochlear nerve fibers andSGNratherthanhaircells.

Spontaneous Proliferation in Organotypic Cultures of Mouse Cochleae

Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regeneration. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse cochlea explants obtained from up to 12 postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine (BrdU) staining and expression of proliferation cell nuclear antigen (PCNA) evaluated with real-time quantitative poly-merase chain reaction(RT-PCR). Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow-down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed.

High-frequency magnetic stimulation attenuates beta-amyloid protein 1-42 neurotoxicity in organotypic hippocampal slices

Repetitive transcranial magnetic stimulation （rTMS） has been utilized as a therapeutic tool for neurodegenerative disorders including Alzheimer＇s disease. However, the precise mechanisms of its clinical effects remain unknown. β-amyloid （Aβ） exhibits direct neurotoxic effects and is closely related to neuronal degeneration in Alzheimer＇s disease. Therefore, it has been hypothesized that the neuroprotective effects of rTMS are related to the mechanisms of protection against Aβ neurotoxicity. Organotypic hippocampal slices were prepared from 8-day old, Sprague Dawley rats. The tissue slices were exposed to 100 μmol/L Al3142 since day 12 in vitro with and without high-frequency （20 Hz） magnetic stimulation. Magnetic stimulation efficacy was evaluated by measuring neuronal nuclei （NeuN） protein expression and by observing cultures following propidium iodide fluorescence staining and bromodeoxyuridine （BrdU） immunohistochemistry. Lactate dehydrogenase activity was detected in the culture media to evaluate hippocampal neuronal damage. Our results demonstrated that high-frequency magnetic stimulation significantly reversed the reduction of NeuN protein expression because of Aβ1-42 exposure （P 〈 0.05） and significantly reduced the number of damaged cells in the hippocampal slices （P 〈 0.05）. However, lactate dehydrogenase levels and anti-BrdU staining results did not reveal any statistical differences These findings indicate that high-frequency magnetic stimulation might have protective effect on hippocampal neurons from Aβ1-42 neurotoxicity.

Pancreatic cancer organotypics: High throughput, preclinical models for pharmacological agent evaluation

Pancreatic cancer carries a terrible prognosis,as the fourth most common cause of cancer death in the Western world.There is clearly a need for new therapies to treat this disease.One of the reasons no effective treatment has been developed in the past decade may in part,be explained by the diverse influences exerted by the tumour microenvironment.The tumour stroma cross-talk in pancreatic cancer can influence chemotherapy delivery and response rate.Thus,appropriate preclinical in vitro models which can bridge simple 2D in vitro cell based assays and complex in vivo models are required to understand the biology of pancreatic cancer.Here we discuss the evolution of 3D organotypic models,which recapitulare the morphological and functional features of pancreatic ductal adenocarcinoma(PDAC).Organotypic cultures are a valid high throughput preclinical in vitro model that maybe a useful tool to help establish new therapies for PDAC.A huge advantage of the organotypic model system is that any component of the model can be easily modulated in a short timeframe.This allows new therapies that can target the cancer,the stromal compartment or both to be tested in a model that mirrors the in vivo situation.A major challenge for the future is to expand the cellular composition of the organotypic model to further develop a system that mimics the PDAC environment more precisely.We discuss how this challenge is being met to increase our understanding of this terrible disease and develop novel therapies that can improve the prognosis for patients.

Role of Nitric Oxide and Nitric Oxide Synthases in Ischemia-reperfusion Injury in Rat Organotypic Hippocampus Slice

To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen glucose deprivation （OGD） for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD （P〈0.05）. The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase （LDH） efflux into the incubation solution. The resuits suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.

Excitotoxic Effects of Glutamate on Cochlear Organotypic Cultures

Glutamate（Glu） is the major afferent excitatory neurotransmitter in the auditory system, and excessive Glu may play an important role in cochlear dysfunction. It is unclear how excessive Glu plays roles in cochlear dysfunction in cochlear organotypic cultures. In this study neonatal rat cochlear organotypic cultures were prepared, and then the cochlear tissues were incubated with a new medium containing specific concentrations of Glu（0.1, 0.5, 1, 10 or 20 mmol/L） for 24 h, or incubated with the medium containing a concentration of 20 mmol/L Glu for 6, 12, 24 or 72 h, respectively. It was found that when the cochlear tissues were cultured for 24 h, the inner hair cells（IHCs） were damaged at the concentration of 0.5 mmol/L Glu, and with the increases of the concentrations, the injury was gradually aggravated, and 20 mmol/L Glu resulted in the significant loss of IHCs. In the 20 mmol/L Glu groups, the stereocilia bundles were missing or disarrayed on a few IHCs after culture for 6 h and the damage effect was time-dependent. The missing of IHCs was more significant in the basal turn of the cochlea than in the middle turn of the cochlea under the same concentration of Glu exposure. These results suggest that excessive exogenous Glu affects the morphology of IHCs, but not affects the outer hair cells（OHCs） in cochlear organotypic cultures, and the excitotoxic effects are different on IHCs of different parts of the cochlea under the same concentration of Glu exposure.

New antioxidant SkQ1 is an effective protector of rat eye retinal pigment epithelium and choroid under conditions of long-term organotypic cultivation

Cells have intrinsic mechanisms for cleaning harmful oxidants represented mainly by reactive oxygen species (ROS). Despite the antioxidant defense, ROS can cause serious damage to the retina that with age leads to various eye diseases and even blindness. Among numerous cell sites of ROS generation, mitochondrial electron transport is of crucial importance. Recently, for the purpose of cleaning ROS in the mitochondrial matrix, powerful mitochondria- targeted antioxidant “SkQ1” has been invented. We studied SkQ1 effects upon tissues of rat posterior eye cup that consisted: retinal pigment epithelium (RPE) ? choroidal coat ? scleral coat. The eye cups were isolated from the eyes of adult albino rats and cultivated in rotary tissue culture system in the presence of 20 nM SkQ1 or without this compound. After 7 days - 1 month in vitro eye cup samples were studied by immunohistochemistry, routine histology, morphometry, and digital image analysis. We have found that under chosen, “in vitro like in vivo” conditions 20 nM SkQ1 effectively reduced cell death in RPE and choroid, protected RPE from disintegration caused by cell phenotypic transformation and withdrawal from the layer, suppressed transmigration of choroidal coat cells. In the ex vivo model we used degenerative processes were more pronounced in the eye cup center where SkQ1 effect was most vivid. All this give us hopes for effectiveness of SkQ1 treatment of retinal central part that is very susceptible to light-induced over-oxidation injury and mostly suffering in many age-related diseases, AMD, in particular.

Transplanted choroidal plexus epithelial cells can integrate with organotypic spinal cord slices into a new system

This study aimed to evaluate the integration of transplanted choroidal plexus epithelial cells with organotypic spinal cord slices.Organotypic spinal cord slices,normally cultured for 6 days,were divided into control group(Ctrl)and transplanted group(T).The choroidal plexus epithelial cells were dissociated and primary cultured(C group).The choroidal plexus epithelial cells cultured for 6–7 days were labeled by 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanineperchlorate(CM-Dil),and were identified by transthyretin(TTR)in immunocytochemistry.They were adjusted to the density of 0.5–1×107/ml,then 2μl cells suspension were transplanted to the spinal cord slices in the T group.The same amount of basal medium was dripped on the spinal cord slices in the Ctrl group.After 14 days of transplantation,the differentiations into neurons and astrocytes,and the synapses were identified by immunofluorescence histochemistry.At the same time,the ratios of cell differentiations and synapses in new system,and the changes of MAPK signaling pathway were tested by western blotting.The choroid plexus epithelial cells were well labeled by CM-Dil and were immune-stained by TTR in immunocytochemistry.The choroid plexus epithelial cells bodies were small when transplanted on the spinal cord slices,but big when transplanted on the polyester membrane inserts.The transplanted cells could differentiate into astrocytes,and possibly differentiate into neurons,and there were a large number of synaptophysin positive vesicles between transplanted cells and organotypic spinal cord slices in immunofluorescence histochemistry.The levels of GFAP,TUB-III and synaptophysin in the T group were higher than which in the Ctrl and C groups in western blotting(P<0.05).And the ratios of p-JNK/JNK and p-P38/P38 in the T group were significantly lower than which in the Ctrl and C groups(P<0.05).But the ratio of p-ERK/ERK in the three groups was of no significant difference.The transplanted choroidal plexus epithelial cells can integrate with organotypic spinal cord slices into a new system.

New antioxidant SkQ1 is an effective protector of rat neural retina under conditions of long-term organotypic cultivation

During life human eye is constantly exposed to sunlight and artificial light, the sources of reactive oxygen species (ROS)—the main cause of age-related eye pathology. A novel mitochondria-targeted antioxidant SkQ1 has recently been invented to reduce mitochondrial ROS by cleaning the mitochondria matrix, “the dirtiest place in the cell” in respect of ROS production and accumulation. Earlier we studied SkQ1 effects upon retinal pigment epithelium and choroid in the rat eye posterior cups exposed to long-term 3D organotypic culturing. It was found that under in vitro conditions 20 nM SkQ1 effectively reduced cell death in retinal pigment epithelium and choroid and protected the tissues from disintegration and cell withdrawal. In the present study we used same ex vivo conditions to examine the effect of SkQ1 upon the rat neural retina kept in the content of the posterior eye cup. Eye cups were isolated and cultured in vitro during 7, 14, and 30 days under rotation in the presence and absence of 20 nM SkQ1 in the culture medium. Serial sections of cultivated eye cups were subjected to histology, computer morphometry and immunohistochemistry. Obtained results show that SkQ1 operates as a strong protective agent, preventing neuronal cell death and other degenerative processes in the neural retina. Cell rescue by SkQ1 was more vivid in the central part of the retina than at the periphery. That, in turn, suggests SkQ1 effectiveness in treatment of some age-related eye diseases when central part of the retina, including macula, is most susceptible to degeneration.

Differentiation of human bone marrow precursor cells into neuronal-like cells after transplantation into canine spinal cord organotypic slice cultures

Background Treatments to regenerate different tissue involving the transplantation of bone marrow derived mesenchymal precursor cells are anticipated. Using an alternative methods, in vitro organotypic slice culture method, would be useful to transplant cells and assessing the effects. This study was to determine the possibility of differentiating human bone marrow precursor cells into cells of the neuronal lineage by transplanting into canine spinal cord organotypic slice cultures. Methods Bone marrow aspirates were obtained from posterior superior iliac spine （PSIS） of patients that had undergone spinal fusion due to a degenerative spinal disorder. For cell imaging, mesenchymal precursor cells （MPCs） were pre-stained with PKH-26 just before transplantation to canine spinal cord slices. Canine spinal cord tissues were obtained from three adult beagle dogs. Spinal cords were cut into transverse slices of 1 mm using tissue chopper. Two slices were transferred into 6-well plate containing 3 ml DMEM with antibiotics. Prepared MPCs （lx104） were transplanted into spinal cord slices. On days 0, 3, 7, 14, MPCs were observed for morphological changes and expression of neuronal markers through immunofluorescence and reverse transcription-polymerase chain reaction （RT-PCR）. Results The morphological study showed： spherical cells in the control and experiment groups on day 0; and on day 3, cells in the control group had one or two thick, short processes and ones in the experiment group had three or four thin, long processes. On day 7, these variously-sized processes contacted each other in the experiment group, but showed typical spindle-shaped cells in the control group. Immunofluorescence showed that PKH-26（＋） MPCs stained positive for NeuN （＋） and GFAP（＋） in experimental group only. Also RT-PCR showed weak expression of β-tubulin III and GFAP. Conclusions Human bone marrow mesenchymal precursor cells （hMPCs） have the potential to differentiate into the neuronal like cells in this canine spinal cord organotypic slice culture model. Furthermore, these findings suggested the possibility that these cells can be utilized to treat patients with spinal cord injuries.

A benchtop brain injury model using resected donor tissue from patients with Chiari malformation

The use of live animal models for testing new therapies for brain and spinal cord repair is a controversial area. Live animal models have associated ethical issues and scientific concerns regarding the predictability of human responses. Alternative models that replicate the 3 D architecture of the central nervous system have prompted the development of organotypic neural injury models. However, the lack of reliable means to access normal human neural tissue has driven reliance on pathological or post-mortem tissue which limits their biological utility. We have established a protocol to use donor cerebellar tonsillar tissue surgically resected from patients with Chiari malformation(cerebellar herniation towards the foramen magnum, with ectopic rather than diseased tissue) to develop an in vitro organotypic model of traumatic brain injury. Viable tissue was maintained for approximately 2 weeks with all the major neural cell types detected. Traumatic injuries could be introduced into the slices with some cardinal features of post-injury pathology evident. Biomaterial placement was also feasible within the in vitro lesions. Accordingly, this ‘proof-of-concept’ study demonstrates that the model offers potential as an alternative to the use of animal tissue for preclinical testing in neural tissue engineering. To our knowledge, this is the first demonstration that donor tissue from patients with Chiari malformation can be used to develop a benchtop model of traumatic brain injury. However, significant challenges in relation to the clinical availability of tissue were encountered, and we discuss logistical issues that must be considered for model scale-up.

Paeonol attenuates inflammation-mediated neurotoxicity and microglial activation

Chronic activation of microglial cells endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. The root of Paeonia lactiflora Pall has been considered useful for the treatment of various disorders in traditional oriental medicine. Paeonol, found in the root of Paeonia lactiflora Pall, has a wide range of pharmacological functions, including anti-oxidative, anti-inflammatory and neuroprotective activities. The objective of this study was to examine the efficacy of paeonol in the repression of inflammation-induced neurotoxicity and microglial cell activation. Organotypic hippocampal slice cultures and primary microglial cells from rat brain were stimulated with bacterial lipopolysaccharide. Paeonol pretreatment was performed for 30 minutes prior to lipopolysaccharide addition. Cell viability and nitrite （the production of nitric oxide）, tumor necrosis factor-alpha and interleukin-lbeta products were measured after lipopolysaccharide treatment. In organotypic hippocampal slice cultures, paeonol blocked lipopolysaccharide-related hippocampal cell death and inhibited the release of nitrite and interleukin-lbeta. Paeonol was effective in inhibiting nitric oxide release from primary microglial cells. It also reduced the lipopolysaccharide-stimulated release of tumor necrosis factor-alpha and intefleukin-1β from microglial cells. Paeonol possesses neuroprotective activity in a model of inflammation-induced neurotoxicity and reduces the release of neurotoxic and proinflammatory factors in activated microglial cells.

Cisplatin-induced vestibular hair cell lesion-less damage at high doses

Cisplatin, a widely used anticancer drug, damages hair cells in cochlear organotypic cultures at low doses, but paradoxically causes little damage at high doses resulting in a U-shaped dose-response function. To determine if the cisplatin dose-response function for vestibular hair cells follows a similar pattern, we treated vestibular organotypic cultures with doses of cisplatin ranging from 10 to 1000 μM. Vestibular hair cell lesions progressively increased as the dose of cisplatin increased with maximum damage occurring around 50-100 μM, but the lesions progressively decreased at higher doses resulting in little hair cell loss at 1000 μM. The U-shaped doseresponse function for cisplatin-treated vestibular hair cells in culture appears to be regulated by copper transporters, Ctrl, ATP7A and ATP7B, that dose-dependently regulate the uptake, sequestration and extrusion of cisplatin.

Biological effect of human serum collected before and after oral intake of Pygeum africanum on various benign prostate cell cultures

Pygeum africanum （Tadenan） is a popular phytotherapeutic agent used in the treatment of symptomatic benign prostatic hyperplasia. The active compounds of the drug have not been identified, and determining the plasma concentration of the drug is, therefore, not possible. Because there are conflicting results on the efficacy of this drug, we aimed to investigate its effect on prostate cell growth in vitro using human serum collected before and after Pygeum africanum intake. We used primary and organotypic cultures of human prostatic stromal myofibroblast cell line WPMY and prostatic epithelial cell line PNT2. We also used fresh benign prostatic tissue. The serum of a treated man induced decreases in the proliferation of primary cells, organotypic cells and WPMY cells but not PNT2 cells. We also analysed the effect of treated serum on the gene expression profile of WPMY cells. The transcriptome analysis revealed an upregulation of genes involved in multiple tumour suppression pathways and a downregulation of genes involved in inflammation and oxidative-stress pathways. The oral intake of Pygeum africanum resulted in serum levels of active substances that were sufficient to inhibit the proliferation of cultured myofibroblasts prostatic cells. This inhibition was associated with changes in the transcriptome. Asian Journal of Andrology（2012） 14, 499-504; doi：lO.1038/aja.2011.132; published online 26 December 2011

Neuroprotective effects of bovine colostrum on intracerebral hemorrhage-induced apoptotic neuronal cell death in rats

Brain cell death after intracerebral hemorrhage may be mediated in part by an apoptotic mechanism Colostrum is the first milk produced by mammals for their young. It plays an important role in protection and development by providing various antibodies, growth factors and nutrients, and has been used for various diseases in many countries. In the present study, we investigated the anti-apoptotic effects of bovine colostrum using organotypic hippocampal slice cultures and an intracerebral hemorrhage animal model. We performed densitometric measurements of propidium iodide uptake, a step-down avoidance task, Nissl staining, and caspase-3 immunohistochemistry. The present results revealed that colostrum treatment significantly suppressed N-methyI-D-aspartic acid-induced neuronal cell death in the rat hippocampus. Moreover, colostrum treatment improved short-term memory by suppressing hemorrhage-induced apoptotic neuronal cell death and decreasing the volume of the lesion induced by intracerebral hemorrhage in the rat hippocampus. These results suggest that colostrum may have a beneficial role in recovering brain function following hemorrhagic stroke by suppressing apoptotic cell death.