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.

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.

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.

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.

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

Teriflunomide provides protective properties after oxygen-glucose-deprivation in hippocampal and cerebellar slice cultures

One of the major challenges in emergency medicine is out-of-hospital cardiac arrest(OHCA).Every year,about 53–62/100000 people worldwide suffer an out-of-hospital cardiac arrest with serious consequences,whereas persistent brain injury is a major cause of morbidity and mortality of those surviving a cardiac arrest.Today,only few and insufficient strategies are known to limit neurological damage of ischemia and reperfusion injury.The aim of the present study was to investigate whether teriflunomide,an approved drug for treatment of relapsing-remitting-multiple-sclerosis,exerts a protective effect on brain cells in an in vitro model of ischemia.Therefore,organotypic slice cultures from rat hippocampus and cerebellum were exposed to oxygen-glucose-deprivation and subsequently treated with teriflunomide.The administration of teriflunomide in the reperfusion time on both hippocampal and cerebellar slice cultures significantly decreased the amount of detectable propidium iodide signal compared with an untreated culture,indicating that more cells survive after oxygen-glucosedeprivation.However,hippocampal slice cultures showed a higher vulnerability to ischemic conditions and a more sensitive response to teriflunomide compared with cerebellar slice cultures.Our study suggests that teriflunomide,applied as a post-treatment after an oxygenglucose-deprivation,has a protective effect on hippocampal and cerebellar cells in organotypic slice cultures of rats.All procedures were conducted under established standards of the German federal state of North Rhine Westphalia,in accordance with the European Communities Council Directive 2010/63/EU on the protection of animals used for scientific purposes.

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.

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.

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.

Seeing the wood for the trees:towards improved quantification of glial cells in central nervous system tissue

The following mini-review attempts to guide researchers in the quantification of fluorescently-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue.It follows from our examination of the utility of Fiji Image J thresholding and binarization algorithms.Describing how we identified the maximum intensity projection as the best of six tested for two dimensional（2 D）-rendering of three-dimensional（3 D） images derived from a series of z-stacked micrographs,the review summarises our comparison of 16 global and 9 local algorithms for their ability to accurately quantify the expression of astrocytic glial fibrillary acidic protein（GFAP）,microglial ionized calcium binding adapter molecule 1（IBA1） and oligodendrocyte lineage Olig2 within fixed cultured rat hippocampal brain slices.The application of these algorithms to chromogenically-stained GFAP and IBA1 within thin tissue sections,is also described.Fiji’s Bio Voxxel plugin allowed categorisation of algorithms according to their sensitivity,specificity accuracy and relative quality.The Percentile algorithm was deemed best for quantifying levels of GFAP,the Li algorithm was best when quantifying IBA expression,while the Otsu algorithm was optimum for Olig2 staining,albeit with over-quantification of oligodendrocyte number when compared to a stereological approach.Also,GFAP and IBA expression in 3,3′-diaminobenzidine（DAB）/haematoxylin-stained cerebellar tissue was best quantified with Default,Isodata and Moments algorithms.The workflow presented in Figure 1 could help to improve the quality of research outcomes that are based on the quantification of protein with brain tissue.

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.

Using a 3-D multicellular simulation of spinal cord injury with live cell imaging to study the neural immune barrier to nanoparticle uptake

Development of nanoparticle （NP） based therapies to promote regeneration in sites of central nervous system （CNS; i.e, brain and spinal cord） pathology relies critically on the availability of experimental models that offer biologically valid predictions of NP fate in vivo. However, there is a major lack of biological models that mimic the pathological complexity of target neural sites in vivo, particularly the responses of resident neural immune cells to NPs. Here, we have utilised a previously developed in vitro model of traumatic spinal cord injury （based on 3-D organotypic slice arrays） with dynamic time lapse imaging to reveal in real-time the acute cellular fate of NPs within injury foci. We demonstrate the utility of our model in revealing the well documented phenomenon of avid NP sequestration by the intrinsic immune cells of the CNS （the microglia）. Such immune sequestration is a known translational barrier to the use of NP-based therapeutics for neurological injury. Accordingly, we suggest that the utility of our model in mimicking microglial sequestration behaviours offers a valuable investigative tool to evaluate strategies to overcome this cellular response within a simple and biologically relevant experimental system, whilst reducing the use of live animal neurological injury models for such studies.

Dissecting brain tumor growth and metastasis in vitro and ex vivo

Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system(CNS)tumors and greatly influence mortality and therapy-induced long-term morbidity in survivors.A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes,to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness.These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments.However,the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel,physiologically more relevant assay systems including organotypic brain slice culture.In this review,using publicly available peer-reviewed primary research and review articles,we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors.Furthermore,we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors.We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors and facilitate the development of more efficacious anti-metastatic treatments.