To begin to explore cell cycle differences in gene expression as one possible cause of RPC heterogeneity, genes that had been identified in other settings as correlated with particular phases of the cell cycle were examined for variations in the single RPC profiles. These genes were divided into two groups, G1/S and G2/M, based upon their reported expression,Hupehenine which has been assayed primarily in cell culture. The heatmap shown in Figure 11A depicts a representative sample of the G1/S group of genes assembled from the literature. Genes such as PCNA, Rrm2 and the Mcms, whose protein products play important roles in DNA replication, were observed in a significant subset of RPCs. Rrm2, for example, was observed in 76% of the profiled RPCs. ISH on retinal cryosections confirmed that these genes were expressed in the ONBL. In the developing retina, the movement of RPCs is coordinated with the cell cycle so that mitosis occurs at the most scleral edge of the retina, just adjacent to the RPE, and S phase occurs toward the vitreal side of the ONBL. The two gap phases, G1 and G2, occur in the intervening space. Closer inspection of the section ISH patterns for Rrm2 and Mcm5 revealed that these genes are more strongly expressed toward the vitreal surface than the scleral surface, indicating they are predominantly detected in S phase cells. At E12.5, the expression pattern of these genes was more scattered Peimisine throughout the ONBL likely reflecting the observation that the precise migration patterns of RPCs with respect to the cell cycle do not occur at this early stage. DISH conducted for Rrm2 showed that 56% of Rrm2+ cells were -thymidine+ at E16.5 and at P0 this number increased to 61%. These data indicate that Rrm2 expression is enriched in S phase cells. However, the microarray data predicted a higher number of cells should express Rrm2 than was observed in the ISH experiments. These results most likely indicate that Rrm2 is expressed in both the G1 and S phases of the cell cycle, but at significantly higher levels in S phase, such that the detection by ISH picks up mostly S phase cells.This would be akin to the situation in serum starved and restimulated fibroblasts where Rrm2 was observed at lower, but detectable, levels during G1, with expression increasing significantly at the G1/S transition and into S phase.

This yielded 94 associated genes whose expression was significantly similar in distribution to cyclin D1. Included in this list were several ribosomal protein genes and other known RPC expressed genes such as Fgf15. The relative expression levels for each of these genes and scaled scores were calculated. Upon inspection, however, the Magnoflorine-iodide distribution of these scores was observed to be very narrow owing to the high levels of expression for many of these genes in the profiled single cells and the persistence of many of these transcripts in newborn neurons. Therefore, to improve the classification of cycling RPCs,Tenacissoside-X additional gene clusters were added to generate a composite RPC score. To generate a composite RPC classification score, three additional genes were chosen to generate gene clusters. These genes have been observed previously in the outer neuroblastic layer of the retina, where the RPCs reside. These 3 genes were also chosen as they together accommodate some of the temporal heterogeneity of the RPCs, as described below. Using the Fisher’s exact test and a cutoff p-value of 1023 as before, associated genes were identified for each of these three genes. The relative expression levels were calculated and scaled RPC scores generated. As shown in Figure 1, 42 cells displayed a significant RPC score. For 36 of these cells, this score was considerably higher than that for RGC, AC, or PR, establishing these single cell profiles as coming from cycling RPCs. These cells are most likely transitional cells, RPCs that are in the process of generating a postmitotic daughter and a full analysis of their gene expression will be presented elsewhere. Since transcripts expressed in RPCs would not be expected to disappear immediately, it was predicted that some cells would possess profiles containing genes expressed in one or more neuronal cell types, together with RPC genes that are in the process of being downregulated. Such transitional cells are of interest as they provide a window into cells that might still be in the process of deciding upon a final fate. If this state was plastic, it might be revealed through the expression of markers of multiple neuronal cell types.

This cell added to the six RPCs designated as transitioning means a total of 7 cells were identified as transitional cells, those having characteristic gene expression of multiple cell types, and these will be discussed in more detail below. In a similar manner to that used for RGCs,Tenacissoside-I classification scores were generated for ACs and PRs using gene clusters built around the transcription factors TCFAP-2b and Nrl respectively. Many of the genes identified as strongly associated with either TCFAP-2b or Nrl were predicted based upon previous work that characterized them as having either AC expression or rod photoreceptor cell expression. In the TCFAP-2b associated genes, at least one previously known AC gene, glycine transporter 1, was not identified because the AC cells isolated in this study were all GABAergic ACs. Using these sets of associated genes to generate classification scores revealed that 4 out of 4 rod photoreceptor cells had significantly higher PR scores than the RGCs and ACs. However, the TCFAP-2b associated genes only yielded Tenacissoside-H considerably higher AC scores for 3 out of the 6 ACs. This result demonstrates the sensitive nature of this classification scheme since it had been previously noted that these single ACs appeared to fall into 2 distinct classes based upon analysis of their gene expression using other methods. Additionally, one of these groups of 3 ACs scored approximately the same for ACs as they did for RGCs. Again, this points to the robust nature of this classification scheme as these cells were also previously observed to have many similarities in gene expression to developing RGCs. Given the success of this classification scheme in sorting out the different types of retinal neurons, it was used to distinguish the profiles of cycling RPCs from those of the developing, but more committed, retinal cell types. Cyclin D1 has been characterized as a gene expressed broadly in cycling RPCs and, therefore, this gene was chosen to generate a list of associated genes for classifying profiled single cells as RPCs. The distribution of cyclin D1 expression was compared pairwise to the signal levels for every other gene on the array across 128 single cell profiles in exactly the same manner as for the RGC, AC and PR markers.

The dosage used for pre-treatment was applied in the study evaluating chronic lithium treatment starting at 22 hpi, and resulted in serum levels of max. 0.22 mmol/l at P35. An increase to 110 mg kg21 d21 did not increase serum concentrations at P35, although lithium serum concentrations of 1.97 mmol/l and 2.64 mmol/l were measured in two infected animals euthanized on P16. An effect of LiCl therapy, at the measured serum concentrations, may be due to the higher lithium serum levels that are reached at a younger age since serum concentrations primarily depend on renal function which is lower in infant rats. This is due to the fact that lithium is not metabolized or Eupalinilide-B bound to any proteins and urinary concentrating ability is fully developed at around 6 weeks of age. Alterations of renal clearance, volume of distribution or other adaptations in the infant rat may be responsible for the different lithium serum concentrations measured at a different age. In the present study in rats that survived PM, lithium led to significantly improved learning performance during probe trials compared to NaCl. The time and distance to reach the platform during training trials decreased in all groups, without reaching statistical significance when comparing animals with LiCl therapy to their littermates receiving NaCl. In earlier studies, chronic lithium treatment improved spatial memory assessed in a water maze during different conditions, Chrysophanol-8-O-β-D-glucopyranoside traumatic brain injury. A correlation between hippocampal neurogenesis and learning has been described earlier. Lithium has been shown to increase neurogenesis, e.g. evidenced by enhanced BrdU labelling of cells in the DG and double-labelling with NeuN. Also, an effect on long-term potentiation has been observed earlier. Immature neurons appear to become involved in spatial memory at 15–20 days of age in rats. Most immature cells die within the first 2 weeks after proliferation, while training during days 6–10 following BrdU injection enhanced survival. PM increases the proliferation of neuronal progenitor cells in the first week after infection with a peak around 2 days post-infection and thereafter declines to basal levels. However, this increase in proliferation does not prevent learning deficits.

In a model of cerebral ischemia, reduction of infarct size and improved neurological outcome after pre-treatment for 16 days with lithium has been described. In the present study, injury to the cortex was only modestly reduced in lithium treated animals. Whether reduced inflammation observed in some other experimental settings is a primary effect of lithium application or results from less injury is unknown. Lithium has been described to act both, pro- and anti-inflammatory and it is known to induce leukocytosis. In the present study,Eupalinilide-D significant up-regulation of TNF, IL-10, and MCP-1 during acute PM was observed in lithium-treated animals, indicating a higher degree of inflammation while IL-1b and IFN-c were down-regulated without reaching statistical significance. Other reports described increasing IL-10 and decreasing TNF concentrations after lithium treatment. However, the effect of lithium on these and other cytokines varied under different conditions. For example, one study reported Eupalinilide-C increased TNF secretion by neutrophils after lithium treatment during an acute inflammatory reaction. In this study, mRNA levels of TNF were not altered, indicating a post-transcriptional regulation. TNF is an early response cytokine triggering an intense immune response and has been targeted in meningitis models as a therapeutic approach. Though, inhibition of TNF activity may be a double-edged sword and interventions aimed at specific immunological mechanisms need to be well balanced. Lithium may increase inflammation locally, evidenced in the present study by raised cyto-/chemokines in CSF samples, while having specific anti-apoptotic properties and preventing hippocampal damage, but not cortical necrosis. In summary, LiCl was able to reduce apoptosis in the hippocampus by favorably modulating the expression of genes involved in the apoptotic machinery. In contrast, LiCl treatment had no impact on weight loss or clinical score. Furthermore, inflammation was not attenuated by LiCl administration and no significant effect on cortical damage could be observed. These proof-of-concept experiments were the basis to investigate the effects of lithium in an adjuvant setting with a clinically relevant treatment regimen.