Screening Of The Differentially Expressed Genes And Cystatin C Expression In The Deafferented Hippocampus

To elucidate the molecular mechanisms underlying the neural plasticity after CNS injury, the gene expression profile in the rat hippocampus following perforant path transections was explored by several methods including custom differential screening, custom cDNA array and cDNA microarray. Of 2300 cDNA clones from low-abundance rat brain cDNA bank, 6 were identified as differentially expressed genes/ESTs in the hippocampus 35 days after lesion, in which none was confirmed by Northern blot. Of 8000 cDNA elements, custom cDNA array identified 47 as differentially expressed genes/ESTs with above 3-fold changes in the hippocampus 35 days after lesion, in which 25 were up-regulated and 22 down-regulated. Northern blot hybridization indicates that false ratio is high with the cDNA array experiment. Of 6234 rat CNS genes/ESTs, cDNA microarray identified 152 as differentially expressed cDNA clones with changes between 1.5-2.8 fold in the hippocampus 10 days following perforant path injury, 32 were up-regulated and 152 down-regulated. Roughly the function categories of the target genes are: immunity reaction, environmental response, cell structure, membrane transport and protein turnover. Other classes include cytokines and their receptors, transcription factors, cell secretion/trafficking, cell adhesion andrecognition and several molecules related to metabolisms. The following Northern blot experiments on a set of randomly picked genes verified the aforementioned cDNA microarray results. These results suggest that the cDNA microarray technique is suitable for studying gene expression profile in large scale in the CNS after injury, and that the present study provides important data for understanding the neural plasticity in the entorhinally deafferented hippocampus.Cystatins are endogenous cysteine protease inhibitors. Along with the target enzymes they modulate the turnover of intracellular and extracellular proteins and are strongly implicated in a variety of animal physiological and pathological processes related to the tissue remodeling. Here we examined the expression of one of the target genes from cDNA array screnning-cystatin C, a major cysteine protease inhibitor of mammalian animals, in the murine hippocampus at 3, 7, 15 and 30 days following perforant path transections. Northern blot analysis showed that cystatin C transcripts were up-regulated in a transient manner, which initiated at 3 days post-lesion, reached maximal levels with a significant increase at 7 and 15 days post-lesion (219 % and 185% of control, respectively), and remained a slight elevation by day 30 post-lesion in the rat hippocampus after entorhinally deafferentation. In situ hybridization and immunohistochemistry confirmed the time-dependent up-regulation of both cystatin C messenger RNA and protein expressions at mouse model. Importantly, the modulation of cystatin C expression was observed to occur specifically in the entorhinally denervated zones: the stratum lacunosum-moleculare of the mouse hippocampus and the outer molecular layer of the dentate gyrus. Double labeling by either combination of in situ hybridization for cystatin C with immunohistochemistry for glial fibrillary acidic protein or double immunofluorescence staining for both proteins in the mousehippocampus at 7 and 15 days post-lesion revealed that most of cystatin C-expressing cells are astrocytes. The role of cystatin C to the neurite outgrowth was subsequently tested by primary neuronal culture. These results suggest that 1. the spatiotemporal up-regulation of cystatin C in the hippocampus is induced by entorhinally deafferentation; 2. cystatin C may be involved in the astroglia-mediated neural plasticity events in the hippocampus following perforant path transections.