BACKGROUND
inflammatory bowel disease , comprising ulcerative colitis and crohn's disease, is characterized by chronic and relapsing inflammation of the gastrointestinal tract.
the pathogenesis of ibd is unknown, but it appears to be multifactorial in origin, and genetic, environmental and dietary factors are believed to be involved  <cit> .
animal models of ibd have been central to the investigation of the pathophysiology of the disease and are valuable tools for drug testing and development.
because ibd-like diseases do not occur spontaneously in animals, several animal models that mimic different aspects of the disease are currently used, including gene knockout, transgenic, chemical, adoptive transfer and spontaneous models  <cit> .
to date, no single model has reproduced all features of the human disease.
one of the most widely used models in both pharmacological and pathophysiological studies  is murine colitis induced by trinitrobenzenesulphonic acid   <cit> .
this simple model is based on a single rectal administration of tnbs dissolved in ethanol.
tnbs is a hapten that elicits an immune response when bound to tissue proteins, while ethanol contributes to disruption of the intestinal barrier.
the result is a severe and prolonged degenerative inflammation of large parts of the colon sharing several clinical and molecular characteristics with crohn's disease.
specifically, the inflammation produced by the administration of tnbs-ethanol involves all layers of the intestinal mucosa and produces long-lasting damage with cell infiltration and ulcers, including protracted physiological dysfunction.
furthermore, both tnbs-ethanol administration to mice and human crohn's disease are characterized by th1-driven inflammation with infiltration of macrophages and neutrophils, producing high levels of proinflammatory cytokines such as tumour necrosis factor, interleukin -1β and il- <dig>  followed by t cell infiltration, mainly of the cd4+ phenotype.
genomic profiling of disease models is of interest for characterizing the pathological response at transcriptome level and identifying putative drug targets.
animal models may overcome many of the limitations of the application of genomic technology to humans, including the need for repeated encoscopy, the large genetic and phenotypic variability, and the difficulty of studying the initial stages of the disease.
there have been a few attempts at gene expression profiling in ibd models  <cit> .
in general, these studies have addressed acute colitis , employed small microarrays , have analysed large samples  and include modest validation experiments .
although a recent study by te velde et al.
<cit>  used  <dig>  transcript microarrays, the data were not validated.
only one of the studies was longitudinal  <cit> .
therefore, the present study represents the most ambitious and comprehensive investigation to date, using several microarrays to examine the progression of colitis at four time points, employing a genechip platform that includes more than  <dig>  transcripts .
results were validated in a subset of almost  <dig> transcripts by using real-time pcr .
the full results database, publicly accessible, will serve as a valuable reference for all researchers in the field.
in fact, three pharmacological studies adopting this strategy are currently underway in our laboratory.
methods
all reagents were obtained from sigma  except where indicated.
animals
female wistar rats  were used, housed in makrolon cages and maintained in air-conditioned animal quarters with a 12-h light-dark cycle.
animals had free access to tap water and were fed a standard chow diet .
this study was carried out in accordance with the directive for the protection of vertebrate animals used for experimental and other scientific purposes of the european union , was approved by the ethical committee of the university of granada and complies with the american physiological society's guiding principles in the care and use of animals.
induction of colitis
colitis was induced as previously described  <cit> .
briefly, rats were fasted overnight and anaesthetized with halothane.
under these conditions, rats were given  <dig> mg of tnbs dissolved in  <dig>  ml of 50% ethanol  by means of a teflon cannula inserted  <dig> cm into the anus.
rats were kept in a head-down position for an additional  <dig> s and returned to their cages.
experimental design
rats were randomly assigned to one of two different groups, a control  group that received a saline enema and a tnbs group  that received the tnbs challenge.
food and water intake and body weight were determined daily.
to follow the progression of the colitis, three rats of the tnbs group were killed  <dig>   <dig>   <dig> and  <dig> days after the induction of colitis.
three control rats were killed on day  <dig> and the other three at the end  of the experiment.
for the purpose of postgenomic validation, this experiment was repeated in order to perform qrt-pcr analysis on fresh samples.
the magnitude and time course of the inflammatory response were similar in both experiments .
assessment of colonic damage
animals were killed by cervical dislocation, and the entire colon was removed and placed on an ice-cold plate, cleaned of fat and mesentery, and blotted on filter paper.
each specimen was weighed and its length measured under a constant load .
the large intestine was longitudinally opened and scored for visible damage on a  <dig> to  <dig> scale as previously described  <cit> .
a sample for genomic analysis was obtained from the distal colon approximately  <dig>  cm proximal to the anus, taking care to avoid any areas of necrosis.
the colon was subsequently divided longitudinally into several pieces for biochemical determinations.
the fragments were immediately frozen in liquid nitrogen and kept at -80°c until used.
myeloperoxidase activity was measured according to the technique described by krawisz et al.
<cit> .
rna extraction, microarray hybridization and data analysis
rna was extracted from homogenized full-thickness colonic tissues in trizol® reagent  and purified with rneasy affinity columns .
quantity and integrity of rna were assessed by spectrophotometry and  <dig> % agarose gel electrophoresis, respectively.
sample labelling, hybridization, staining and scanning procedures were carried out using affymetrix standard protocols .
the microarray analysis was performed by progenika biopharma  on  <dig> genechip® rat expression array  <dig>  <dig>  microchips .
normalization and statistical analyses were carried out using genespring v <dig>  .
gene ontology analysis was performed with genemapp/mappfinder  <cit> .
specifically, biological process, cellular component and molecular function categories were scored with respect to the number of genes included in the rattus norvegicus database that were measured in the microarray and the number of genes significantly affected by tnbs colitis in each category.
the resulting z parameter  <cit>  has positive values when the proportion of genes affected is higher than expected, and it was used to select the most representative gene ontology categories  at each time point.
non-redundant categories are shown, listing only the highest z value when nested categories exceeded the cutoff value at multiple levels.
the k-means algorithm was applied to identify and group transcript changes over time in clusters.
only sequences with annotated gene identities that were present  in at least 66% of samples per group in at least one group were considered.
the data were analyzed by analysis of variance followed by tukey post-hoc tests in order to reduce the false positive or type i error rate in inter-group comparisons.
in addition, the benjamini & hochberg false discovery rate correction was applied to reduce the occurrence of type i error when comparing among genes.
this procedure offers a reasonable balance between sensitivity and specificity.
data are expressed as fold change  over the control  group  at each time point .
miame recommendations  <cit>  were followed to ensure that all information needed to understand, interpret, reproduce and compare our results was given in detail.
the data are accessible at the the european bioinformatics institute arrayexpress database .
postgenomic validation
postgenomic validation was carried out by measuring  <dig> of the genes in fresh samples  using qrt-pcr with taqman® low density arrays .
genes were selected to include both significantly and non-significantly changed genes pertaining to different families affected by inflammation, i.e., those related to transport, immunity or metabolism.
the relative ct values of each gene with respect to the reference gene  were used to calculate the rq  parameter, which represents the change in mrna expression compared to a control sample.
the rq was then used to calculate fold change ratios.
results are expressed as mean ± sem.
RESULTS
tnbs colitis
the morphological and biochemical features of tnbs colitis were consistent with previous reports by our group and other authors  <cit> .
thus, tnbs-treated rats suffered anorexia and loss of body weight  associated with extensive mucosal damage, oedema, haemorrhage and early epithelial necrosis.
leukocyte infiltration was prominent, resulting in a significant increase in myeloperoxidase activity .
epithelial regeneration gradually occurred from day  <dig> and was macroscopically complete by day  <dig>  at  <dig> and  <dig> days, there was major submucosal fibrosis and scarring that resulted in a marked shortening of colonic length.
even after  <dig> weeks, treated rats showed significant differences with controls in colonic weight-to-length ratio and myeloperoxidase activity, among others .
genomic analysis
<dig> genes were significantly modified at  <dig> days after tnbs instillation .
this number was almost doubled at day  <dig>  and rose to  <dig>  by day  <dig>  the pool of altered gene expression fell to  <dig> by day  <dig> , coinciding with a marked recovery from the inflammatory bout.
the dendogram in figure  <dig> shows that samples from tnbs colitic rats at each time point were clustered together, forming groups that significantly differed from control samples.
as expected, many of the genes that showed significant changes are directly related to the inflammatory response, including genes for chemokines, cytokines and inflammatory markers such as cp, ptgs <dig>  or lyz .
an additional set of three rats per experimental group was subjected to confirmatory analysis of  <dig> genes by qrt-pcr.
this independent validation procedure for microarray analysis results has become standard in genomic studies, although our use of triplicate measurements may have made this step redundant.
an excellent correlation was found between microarray and qrt-pcr data .
individual examples can be seen in other figures .
amplification was not possible with two of the selected genes, namely defb <dig> and htr <dig>  in addition, neither il <dig> nor ifng could be detected in the control samples but both were measured in the colitis groups.
hence, although this is an unmistakable indication of significant induction, a fold change could not be calculated from the qrt-pcr data.
the reason for this discrepancy is not known.
non-redundant categories with z values >  <dig> are shown.
a high z value indicates a higher representation of significantly changed genes than expected.
time course analysis
the set of genes that was significantly altered by tnbs colitis underwent k-means clustering, a mathematical tool that groups genes with a similar time evolution profile.
most genes clustered to profiles that showed the highest increase  at  <dig> and  <dig> days, usually peaking at  <dig> days or staying relatively constant between  <dig> and  <dig> days and decreasing or increasing at  <dig> days .
however, some atypical profiles were also detected.
thus, the expression of some genes was changed only at day  <dig>  or day  <dig>  while others were upregulated  or downregulated  at all or most time points.
therefore, somewhat unexpectedly, the time course of gene expression was largely parallel, with few outliers, although there were marked differences in the magnitude of changes.
however, as explained above, there was a change over time in the clustering of gene groups according to gene ontology.
only annotated genes with a 50% or higher fold change are included.
in all cases the fc at days 5– <dig> was not significantly different from the control .
only annotated genes with a 50% or higher fold change are included.
in all cases the fc at days  <dig> –  <dig> was not significantly different from the control .
genes involved in the inflammatory response
many of the most severely affected genes were those directly involved in the inflammatory response, as expected .
chemokines were especially prominent, including neutrophil chemokines and other leukocyte-attractant molecules.
interleukin  <dig> and related tlr <dig> pathways were highly affected, and both converged in the activation of nf-κb.
nfkbia  was markedly increased, indicating a trend to limit activation of this pathway.
the intestinal inflammatory marker alpl  <cit>  was prominently upregulated, unlike alpi and alpi <dig>  the intestinal isoforms of alkaline phosphatase .
the prostaglandin biosynthetic pathway was strongly activated by tnbs through the concerted induction of pla2g2a, ptgs <dig>  and ptges, especially during the chronic phase .
other affected genes are related to cell adhesion, bacteria binding or reactive oxygen species generation.
there were also marked changes in the expression of numerous transcripts involved in antioxidative defence, most of which were downregulated.
in addition, close examination of the expression level of a number of genes that act as inflammatory cell markers indicated the nature of the inflammatory infiltrate.
thus, tnbs colitis was characterized by an absence of significant changes in markers of t cells , b cells  and nk cells .
in contrast, several neutrophil/macrophage markers such as s100a <dig>  itgb <dig> , cd68_predicted, csf1r, cybb, csf2rb <dig> and lcn <dig> were significantly increased by inflammation, especially at days  <dig> and  <dig>
genes involved in metabolism
metabolism-related genes were highly affected  by tnbs-induced colitis .
they included genes participating in glycolysis, like aldob and many different subunits of the pyruvate dehydrogenase complex .
several enzymes of the krebs cycle were also changed, e.g., idh3g, aco <dig>  sucla <dig> and cs.
the respiratory chain genes ndufs <dig> and sdhc and several genes encoding cytochrome isoforms were also downregulated.
with regard to lipid metabolism, the fatty acid biosynthetic enzymes fasn and scd <dig> were inhibited, as was the related gene acly.
several transcripts related to cholesterol biosynthesis and transport were also affected .
in addition, both apoe and the apolipoprotein b mrna editing gene apobec <dig> were upregulated, suggesting a higher lipoprotein biosynthesis, although apoe may also play a protective role against lipopolysaccharide  <cit> .
a number of other metabolic pathways appeared to be altered by tnbs colitis, including purine de novo biosynthesis, arginine and heme catabolism.
genes involved in signalling
many transcripts involved in cell signalling were changed by tnbs colitis, including genes encoding proteins that participate in camp/protein kinase a and calcium/protein kinase c pathways, phosphatases, various regulatory proteins and a number of transmitter/hormone receptors, including p2ry <dig>  htr2b, prlr, sstr <dig>  tacr <dig> and thra.
in addition, ace was modestly increased only on day  <dig>  suggesting a possible local increase in angiotensin ii production in the healing colonic tissue .
the signalling-related genes that exhibited the highest changes in expression included gp49b and plek.
gp49b encodes an immunoglobulin-like receptor expressed in myeloid cells that appears to counter-regulate the cytokine and chemokine attraction of neutrophils  <cit> .
the function of plek appears to be related to the regulation of macrophage phagocytosis  <cit> .
genes involved in transport
tnbs colitis influenced the expression of many transport-related transcripts, including genes that participate in ionic transport, e.g.
slc9a <dig>  slc9a <dig> , scnn1a and slc26a <dig> , which mediate nacl absorption, and the na+/k+ pump subunit atp1a <dig> and slc12a <dig> , all of which were inhibited.
however, the chloride channel cftr was not affected.
three aquaporins  were also repressed in tnbs colitis, whereas aqp <dig> was upregulated.
many other nonionic transporters and ionic channels were altered .
interestingly, there was upregulation of slc7a <dig>  which is involved in the basolateral transport of arginine, lysine and ornithine by epithelial cells and activated monocytes  <cit> , suggesting increased availability of inos substrate.
genes involved in tissue remodelling
following the gene ontology analysis, a manual search of the most severely affected genes confirmed the dramatic changes in the expression of many genes involved in matrix deposition, muscle plasticity and angiogenesis .
these include igf <dig>  and igfbp <dig>  genes that may regulate tissue remodelling by increasing collagen synthesis and cell proliferation.
several procollagen/collagen isoforms were increased, as well as genes involved in collagen processing and synthesis or in collagen and elastin fibre cross-linking .
a number of metalloproteases and multiple cytoskeletal genes were increased, especially during the chronic phase.
discussion
the main features of the tnbs model of colitis have been well defined.
ethanol causes direct toxic damage to the epithelium, which in turn grants tnbs access to the colonic mucosa, where it acts as a hapten.
although the toxic effect of ethanol is a requisite for tnbs to act effectively, the immunogenic nature of the 'chronic' phase has been unequivocally established by various authors  <cit> .
without tnbs, ethanol produces only a short-lived inflammatory reaction that resolves without sequelae and was therefore not of interest in the present study.
during the 'chronic' phase, tnbs rat colitis shares a number of features in common with ibd, e.g., transmural inflammation , abnormal ion transport, diarrhoea, fibrosis and abnormal intestinal motility.
furthermore, tnbs rat colitis is amenable to treatment with corticoids, sulfasalazine or tacrolimus, making it an attractive model for the preclinical testing of putative ibd drugs.
our aim was to perform a detailed genomic characterization of the model.
the tnbs colonic inflammatory response was characterized by a marked increase in the expression of multiple genes involved in inflammation/immunity, including cytokines, chemokines, adhesion molecules, eicosanoid-related genes, and a number of effectors and cell markers.
there was a dramatic increase in il1b, il1a, il <dig>  tgfb <dig> and tnf and in various chemokine and chemokine receptors.
in addition, a nonsignificant increase was observed in the pivotal il23a and ifng cytokine genes, which was found to be significant after re-examination using the more sensitive qrt-pcr.
although no protein data are avaliable, it is tempting to speculate that the combined upregulation of il23a, il <dig> and tgfb <dig> are indicative of th <dig> induction  <cit> .
in fact, although il17a was not represented in the microarray, qrt-pcr findings showed an increase in the mrna levels , suggesting the involvement of th <dig> cells in rat tnbs colitis.
a contribution of th <dig> cells is also likely in view of the ifng upregulation and lack of il <dig> changes, although il12a and il12b were not augmented.
our group  <cit>  and other authors  <cit>  previously reported an increase in the ifn-γ or ifn-γ/il- <dig> protein ratio in this model.
a staggering increase was observed in some of the genes, e.g.
s100a9/s100a <dig>  suggesting that they may be highly sensitive markers to follow intestinal inflammation, as previously observed in humans  <cit> .
these genes encode proteins expressed by macrophages and neutrophils and are involved in bacterial defence, chemotaxis and signalling  <cit> .
analysis of the microarray data offered insights into the nature of the inflammatory infiltrate.
tnbs colitis was previously characterized at this level  <cit> .
in brief, macrophages and neutrophils are initially recruited to the colonic mucosa and submucosa, followed by a more predominant role of lymphocytes in later stages of colitis, which are characterized by progressive healing, reepithelialization, crypt enlargement and prominent fibrosis  <cit> .
the predominance of neutrophils/macrophages in the inflammatory response was confirmed in our study by the global increase in gene markers of these cell types and the absence of effects on b, t and nk cells .
it should be noted that the sample used for genomic analysis did not include lymphoid follicles, in which lymphocytes accumulate in this model  <cit> , which explains the lack of a significant increase.
however, gene ontology analysis indicated that lymphocyte differentiation was already prominent at day  <dig>
our data also revealed important changes that were not directly linked to inflammation/immunity.
thus, marked alterations were found in metabolism-related genes, indicating a reduced functionality of numerous biochemical pathways, including glycolysis, purine biosynthesis, krebs cycle, cholesterol biosynthesis and transport, fatty acid biosynthesis and respiratory chain.
taken together, these data demonstrate a decrease in biosynthesis of macromolecules, an increase in catabolism, and a decrease in aerobic and anaerobic respiration.
therefore, mucosal cells may be have inadequate energy resources during the inflammatory response.
similar changes have been reported in ibd patients  <cit>  and in experimental colitis  <cit> .
a number of genes encoding ion transporters were affected by tnbs colitis, including: atp1a <dig>  encoding the catalytic subunit of the na+/k+ pump; scnn1a, encoding the alpha polypeptide of the epithelial sodium channel; nhe <dig>  encoding the housekeeping na+/h+ exchanger; and slc12a <dig>  encoding nkcc <dig>  the na+/k+/cl- cotransporter.
these were generally downregulated, as were the transporters involved in nacl absorption, e.g., nhe <dig>  nhe <dig>  and slc26a <dig>  however, cftr was unchanged.
these modifications are consistent with severe alterations of ionic transport, as demonstrated in ibd and animal models  <cit> .
a striking finding of our analysis was the vast number of upregulated genes involved in tissue remodelling in tnbs colitis, generally during the chronic phase, as indicated by the gene ontology analysis.
the chronic phase is characterized by mucosal wound healing and submucosal fibrosis and by the scarring and deposition of excess muscular tissue, extending through day  <dig> and eventually leaving sequelae  <cit> .
these features are often detectable by the naked eye as colonic shortening, deformation and rigidity.
two features are related to the intestinal fibrosis suffered by ibd patients: an increase in collagen synthesis by smooth muscle cells, fibroblasts and myofibroblasts, and an increase in muscle layer thickness.
igfbp- <dig> expression is known to increase in ulcerative colitis, while igf- <dig> and tgf-β <dig> expression is known to increase in both ulcerative colitis and crohn's disease  <cit> .
these molecules have been related to extracellular matrix remodelling and may therefore be relevant to the fibrosis observed in ibd.
our results are in agreement with these ibd findings, showing an increase in igf <dig>  igfbp <dig> and tgfb <dig> expression in the inflamed colon.
hence, the tnbs model may be appropriate for the study of molecular mechanisms implicated in the development of fibrosis in intestinal inflammation.
in addition, expressions of procollagen/collagen genes, most notably tgm <dig>  and of several enzymes related to collagen processing were increased in the tnbs colitic animals.
our results include some intriguing findings, including: the severe repression of nuclear receptors nr1d <dig> and nr1d <dig>  involved in circadian expression patterns and recently shown to be regulated by heme  <cit> ; the synthesis and marked upregulation of tg in the inflamed colon; and the downregulation of the thyroid receptor.
further experiments are warranted to clarify the role of these changes in colonic inflammation.
we were especially interested in establishing a correlation between the known characteristics of the model and the time course of gene expression.
because the time points selected cover all stages of the tnbs-induced inflammatory response, from acute  to chronic  and healing/recovery  phases, we expected to find substantial differences in the pattern of expression.
for instance, genes involved in inflammation/immunity and tissue regeneration were expected to predominate in the early and late stages, respectively.
however, the vast majority of transcripts followed a common trend, namely a change of expression that was maximal at days  <dig> and  <dig>  when colitis is most prominent, and was normal  at day  <dig>  hence, there is a close correlation between the pathological features of tnbs colitis and the changes observed in the transcriptome.
the genes that were up- or downregulated only at days  <dig> or  <dig> did not conform to any specific category.
some of the transcripts involved are of interest.
thus, retnla, which encodes resistin-like alpha, which is dramatically repressed soon after colitis induction, may be involved in monocyte activation, as is the beta isoform  <cit> .
both ace and cma <dig>  which encode enzymes with angiotensin i cleaving capacity, are induced specifically at day  <dig>  some specific receptors involved in cellular signalling are differentially regulated, such as galr <dig>  hrh <dig>  npffr <dig> and cnr <dig>  further analysis is warranted to explore the implications of these findings.
despite using a technical approach  that meets or surpasses common standards in genomic studies, we carried out more than  <dig> qrt-pcr determinations in  <dig> additional samples for a posteriori data validation.
as expected, the overall correlation was excellent.
in some cases, microarray analysis appeared to be less prone to pick up statistically significant variations, i.e.
it was less sensitive than qrt-pcr.
the fact that we examined four different time points adds to the complexity of the analysis, since a marked change at one time point may not be enough in many cases to reach significance in the anova.
further validation is provided by the fact that the changes  observed in many genes, including il1b, il1rn, cftr, tnf, alpl, alpi, ifng, il <dig>  il <dig>  pgts <dig>  nos <dig> and ccl <dig>  among many others, are in line with previous observations from our own group, either at the rna or protein level  <cit> .
previous studies have examined changes in the transcriptome in intestinal inflammation, especially in mouse colitis elicited by tnbs and other manoeuvres  <cit> .
these studies are somewhat limited because they either use small microarrays, examine animals shortly after colitis induction , offer limited or no postgenomic validation or use longitudinal tissue samples.
one of these studies addressed the time course of gene expression, but it studied recovery from repeated tnbs challenges  <cit> .
in general, the changes in gene expression documented in the present study are much stronger and wider than those found in previous reports.
for example,  <dig> genes were differentially expressed in the above study in compared to the thousands of genes in our investigation.
on the other hand, there have been several human ibd microarray studies  <cit> , which have detected some key gene expression changes associated with human intestinal inflammation, including some specifically linked to either ulcerative colitis or crohn's disease  <cit> .
however, it is poorly understood how genomic data are related to ibd pathology and therapy, partly because of the variability inherent to human studies and partly because of methodological differences  <cit> .
for instance, the most important source of variation in gene expression measurement  is the microarray platform itself  <cit> , and all of the above studies used different arrays.
there are also differences in design and especially in sample selection, i.e., individual vs. pooled or mucosal vs. full thickness.
nevertheless, there are some similarities with the rat tnbs model, including increases in chi3l <dig>  mmp <dig>   <dig> and  <dig>  apoe, s100a <dig>  ltb, sod <dig>  cxcl <dig>  cxcl <dig>  il <dig> and many other genes.
CONCLUSIONS
we have characterized the rat tnbs model of ibd at the genomic level, obtaining novel data on individual genes, metabolic pathways and biological functions that are altered during colitis.
these findings establish a basis for further mechanistic studies on drug action and research into the pathophysiology of intestinal inflammation.
the full genomic database will serve as a reference standard for all future investigations using this model and for comparisons with other animal models of colitis, an approach that is currently being applied in our laboratory.
abbreviations
ibd: inflammatory bowel disease; il: interleukin; qrt-pcr: quantitative real time pcr; tnbs: trinitrobenzenesulphonic acid.
authors' contributions
fsmlh and oma conducted the animal experiments, coordinated the bioinformatic analysis of the microarray and qrt-pcr data and drafted the manuscript.
fsmlh, oma, mds and az conceived the study.
all authors contributed to the interpretation of the data and have read and approved the final manuscript.

 inflammatory bowel disease, comprising ulcerative colitis and crohn's disease, is characterized by chronic and relapsing inflammation of the gastrointestinal tract. the pathogenesis of ibd is unknown, but it appears to be multifactorial in origin, and genetic, environmental and dietary factors are believed to be involved. animal models of ibd have been central to the investigation of the pathophysiology of the disease and are valuable tools for drug testing and development. because ibd-like diseases do not occur spontaneously in animals, several animal models that mimic different aspects of the disease are currently used, including gene knockout, transgenic, chemical, adoptive transfer and spontaneous models. to date, no single model has reproduced all features of the human disease. one of the most widely used models in both pharmacological and pathophysiological studies is murine colitis induced by trinitrobenzenesulphonic acid. this simple model is based on a single rectal administration of tnbs dissolved in ethanol. tnbs is a hapten that elicits an immune response when bound to tissue proteins, while ethanol contributes to disruption of the intestinal barrier. the result is a severe and prolonged degenerative inflammation of large parts of the colon sharing several clinical and molecular characteristics with crohn's disease. specifically, the inflammation produced by the administration of tnbs-ethanol involves all layers of the intestinal mucosa and produces long-lasting damage with cell infiltration and ulcers, including protracted physiological dysfunction. furthermore, both tnbs-ethanol administration to mice and human crohn's disease are characterized by th1-driven inflammation with infiltration of macrophages and neutrophils, producing high levels of proinflammatory cytokines such as tumour necrosis factor, interleukin -1β and il- followed by t cell infiltration, mainly of the cd4+ phenotype. genomic profiling of disease models is of interest for characterizing the pathological response at transcriptome level and identifying putative drug targets. animal models may overcome many of the limitations of the application of genomic technology to humans, including the need for repeated encoscopy, the large genetic and phenotypic variability, and the difficulty of studying the initial stages of the disease. there have been a few attempts at gene expression profiling in ibd models. in general, these studies have addressed acute colitis, employed small microarrays, have analysed large samples and include modest validation experiments. although a recent study by te velde et al. used transcript microarrays, the data were not validated. only one of the studies was longitudinal. therefore, the present study represents the most ambitious and comprehensive investigation to date, using several microarrays to examine the progression of colitis at four time points, employing a genechip platform that includes more than transcripts. results were validated in a subset of almost transcripts by using real-time pcr. the full results database, publicly accessible, will serve as a valuable reference for all researchers in the field. in fact, three pharmacological studies adopting this strategy are currently underway in our laboratory. methods all reagents were obtained from sigma except where indicated. animals female wistar rats were used, housed in makrolon cages and maintained in air-conditioned animal quarters with a 12-h light-dark cycle. animals had free access to tap water and were fed a standard chow diet. this study was carried out in accordance with the directive for the protection of vertebrate animals used for experimental and other scientific purposes of the european union, was approved by the ethical committee of the university of granada and complies with the american physiological society's guiding principles in the care and use of animals. induction of colitis colitis was induced as previously described. briefly, rats were fasted overnight and anaesthetized with halothane. under these conditions, rats were given mg of tnbs dissolved in ml of 50% ethanol by means of a teflon cannula inserted cm into the anus. rats were kept in a head-down position for an additional s and returned to their cages. experimental design rats were randomly assigned to one of two different groups, a control group that received a saline enema and a tnbs group that received the tnbs challenge. food and water intake and body weight were determined daily. to follow the progression of the colitis, three rats of the tnbs group were killed and days after the induction of colitis. three control rats were killed on day and the other three at the end of the experiment. for the purpose of postgenomic validation, this experiment was repeated in order to perform qrt-pcr analysis on fresh samples. the magnitude and time course of the inflammatory response were similar in both experiments. assessment of colonic damage animals were killed by cervical dislocation, and the entire colon was removed and placed on an ice-cold plate, cleaned of fat and mesentery, and blotted on filter paper. each specimen was weighed and its length measured under a constant load. the large intestine was longitudinally opened and scored for visible damage on a to scale as previously described. a sample for genomic analysis was obtained from the distal colon approximately cm proximal to the anus, taking care to avoid any areas of necrosis. the colon was subsequently divided longitudinally into several pieces for biochemical determinations. the fragments were immediately frozen in liquid nitrogen and kept at -80°c until used. myeloperoxidase activity was measured according to the technique described by krawisz et al.. rna extraction, microarray hybridization and data analysis rna was extracted from homogenized full-thickness colonic tissues in trizol® reagent and purified with rneasy affinity columns. quantity and integrity of rna were assessed by spectrophotometry and % agarose gel electrophoresis, respectively. sample labelling, hybridization, staining and scanning procedures were carried out using affymetrix standard protocols. the microarray analysis was performed by progenika biopharma on genechip® rat expression array microchips. normalization and statistical analyses were carried out using genespring v. gene ontology analysis was performed with genemapp/mappfinder. specifically, biological process, cellular component and molecular function categories were scored with respect to the number of genes included in the rattus norvegicus database that were measured in the microarray and the number of genes significantly affected by tnbs colitis in each category. the resulting z parameter has positive values when the proportion of genes affected is higher than expected, and it was used to select the most representative gene ontology categories at each time point. non-redundant categories are shown, listing only the highest z value when nested categories exceeded the cutoff value at multiple levels. the k-means algorithm was applied to identify and group transcript changes over time in clusters. only sequences with annotated gene identities that were present in at least 66% of samples per group in at least one group were considered. the data were analyzed by analysis of variance followed by tukey post-hoc tests in order to reduce the false positive or type i error rate in inter-group comparisons. in addition, the benjamini & hochberg false discovery rate correction was applied to reduce the occurrence of type i error when comparing among genes. this procedure offers a reasonable balance between sensitivity and specificity. data are expressed as fold change over the control group at each time point. miame recommendations were followed to ensure that all information needed to understand, interpret, reproduce and compare our results was given in detail. the data are accessible at the the european bioinformatics institute arrayexpress database. postgenomic validation postgenomic validation was carried out by measuring of the genes in fresh samples using qrt-pcr with taqman® low density arrays. genes were selected to include both significantly and non-significantly changed genes pertaining to different families affected by inflammation, i.e., those related to transport, immunity or metabolism. the relative ct values of each gene with respect to the reference gene were used to calculate the rq parameter, which represents the change in mrna expression compared to a control sample. the rq was then used to calculate fold change ratios. results are expressed as mean ± sem.  tnbs colitis the morphological and biochemical features of tnbs colitis were consistent with previous reports by our group and other authors. thus, tnbs-treated rats suffered anorexia and loss of body weight associated with extensive mucosal damage, oedema, haemorrhage and early epithelial necrosis. leukocyte infiltration was prominent, resulting in a significant increase in myeloperoxidase activity. epithelial regeneration gradually occurred from day and was macroscopically complete by day at and days, there was major submucosal fibrosis and scarring that resulted in a marked shortening of colonic length. even after weeks, treated rats showed significant differences with controls in colonic weight-to-length ratio and myeloperoxidase activity, among others. genomic analysis genes were significantly modified at days after tnbs instillation. this number was almost doubled at day and rose to by day the pool of altered gene expression fell to by day, coinciding with a marked recovery from the inflammatory bout. the dendogram in figure shows that samples from tnbs colitic rats at each time point were clustered together, forming groups that significantly differed from control samples. as expected, many of the genes that showed significant changes are directly related to the inflammatory response, including genes for chemokines, cytokines and inflammatory markers such as cp, ptgs or lyz. an additional set of three rats per experimental group was subjected to confirmatory analysis of genes by qrt-pcr. this independent validation procedure for microarray analysis results has become standard in genomic studies, although our use of triplicate measurements may have made this step redundant. an excellent correlation was found between microarray and qrt-pcr data. individual examples can be seen in other figures. amplification was not possible with two of the selected genes, namely defb and htr in addition, neither il nor ifng could be detected in the control samples but both were measured in the colitis groups. hence, although this is an unmistakable indication of significant induction, a fold change could not be calculated from the qrt-pcr data. the reason for this discrepancy is not known. non-redundant categories with z values > are shown. a high z value indicates a higher representation of significantly changed genes than expected. time course analysis the set of genes that was significantly altered by tnbs colitis underwent k-means clustering, a mathematical tool that groups genes with a similar time evolution profile. most genes clustered to profiles that showed the highest increase at and days, usually peaking at days or staying relatively constant between and days and decreasing or increasing at days. however, some atypical profiles were also detected. thus, the expression of some genes was changed only at day or day while others were upregulated or downregulated at all or most time points. therefore, somewhat unexpectedly, the time course of gene expression was largely parallel, with few outliers, although there were marked differences in the magnitude of changes. however, as explained above, there was a change over time in the clustering of gene groups according to gene ontology. only annotated genes with a 50% or higher fold change are included. in all cases the fc at days 5– was not significantly different from the control. only annotated genes with a 50% or higher fold change are included. in all cases the fc at days – was not significantly different from the control. genes involved in the inflammatory response many of the most severely affected genes were those directly involved in the inflammatory response, as expected. chemokines were especially prominent, including neutrophil chemokines and other leukocyte-attractant molecules. interleukin and related tlr pathways were highly affected, and both converged in the activation of nf-κb. nfkbia was markedly increased, indicating a trend to limit activation of this pathway. the intestinal inflammatory marker alpl was prominently upregulated, unlike alpi and alpi the intestinal isoforms of alkaline phosphatase. the prostaglandin biosynthetic pathway was strongly activated by tnbs through the concerted induction of pla2g2a, ptgs and ptges, especially during the chronic phase. other affected genes are related to cell adhesion, bacteria binding or reactive oxygen species generation. there were also marked changes in the expression of numerous transcripts involved in antioxidative defence, most of which were downregulated. in addition, close examination of the expression level of a number of genes that act as inflammatory cell markers indicated the nature of the inflammatory infiltrate. thus, tnbs colitis was characterized by an absence of significant changes in markers of t cells, b cells and nk cells. in contrast, several neutrophil/macrophage markers such as s100a itgb, cd68_predicted, csf1r, cybb, csf2rb and lcn were significantly increased by inflammation, especially at days and genes involved in metabolism metabolism-related genes were highly affected by tnbs-induced colitis. they included genes participating in glycolysis, like aldob and many different subunits of the pyruvate dehydrogenase complex. several enzymes of the krebs cycle were also changed, e.g., idh3g, aco sucla and cs. the respiratory chain genes ndufs and sdhc and several genes encoding cytochrome isoforms were also downregulated. with regard to lipid metabolism, the fatty acid biosynthetic enzymes fasn and scd were inhibited, as was the related gene acly. several transcripts related to cholesterol biosynthesis and transport were also affected. in addition, both apoe and the apolipoprotein b mrna editing gene apobec were upregulated, suggesting a higher lipoprotein biosynthesis, although apoe may also play a protective role against lipopolysaccharide. a number of other metabolic pathways appeared to be altered by tnbs colitis, including purine de novo biosynthesis, arginine and heme catabolism. genes involved in signalling many transcripts involved in cell signalling were changed by tnbs colitis, including genes encoding proteins that participate in camp/protein kinase a and calcium/protein kinase c pathways, phosphatases, various regulatory proteins and a number of transmitter/hormone receptors, including p2ry htr2b, prlr, sstr tacr and thra. in addition, ace was modestly increased only on day suggesting a possible local increase in angiotensin ii production in the healing colonic tissue. the signalling-related genes that exhibited the highest changes in expression included gp49b and plek. gp49b encodes an immunoglobulin-like receptor expressed in myeloid cells that appears to counter-regulate the cytokine and chemokine attraction of neutrophils. the function of plek appears to be related to the regulation of macrophage phagocytosis. genes involved in transport tnbs colitis influenced the expression of many transport-related transcripts, including genes that participate in ionic transport, e.g. slc9a slc9a, scnn1a and slc26a, which mediate nacl absorption, and the na+/k+ pump subunit atp1a and slc12a, all of which were inhibited. however, the chloride channel cftr was not affected. three aquaporins were also repressed in tnbs colitis, whereas aqp was upregulated. many other nonionic transporters and ionic channels were altered. interestingly, there was upregulation of slc7a which is involved in the basolateral transport of arginine, lysine and ornithine by epithelial cells and activated monocytes, suggesting increased availability of inos substrate. genes involved in tissue remodelling following the gene ontology analysis, a manual search of the most severely affected genes confirmed the dramatic changes in the expression of many genes involved in matrix deposition, muscle plasticity and angiogenesis. these include igf and igfbp genes that may regulate tissue remodelling by increasing collagen synthesis and cell proliferation. several procollagen/collagen isoforms were increased, as well as genes involved in collagen processing and synthesis or in collagen and elastin fibre cross-linking. a number of metalloproteases and multiple cytoskeletal genes were increased, especially during the chronic phase. discussion the main features of the tnbs model of colitis have been well defined. ethanol causes direct toxic damage to the epithelium, which in turn grants tnbs access to the colonic mucosa, where it acts as a hapten. although the toxic effect of ethanol is a requisite for tnbs to act effectively, the immunogenic nature of the'chronic' phase has been unequivocally established by various authors. without tnbs, ethanol produces only a short-lived inflammatory reaction that resolves without sequelae and was therefore not of interest in the present study. during the'chronic' phase, tnbs rat colitis shares a number of features in common with ibd, e.g., transmural inflammation, abnormal ion transport, diarrhoea, fibrosis and abnormal intestinal motility. furthermore, tnbs rat colitis is amenable to treatment with corticoids, sulfasalazine or tacrolimus, making it an attractive model for the preclinical testing of putative ibd drugs. our aim was to perform a detailed genomic characterization of the model. the tnbs colonic inflammatory response was characterized by a marked increase in the expression of multiple genes involved in inflammation/immunity, including cytokines, chemokines, adhesion molecules, eicosanoid-related genes, and a number of effectors and cell markers. there was a dramatic increase in il1b, il1a, il tgfb and tnf and in various chemokine and chemokine receptors. in addition, a nonsignificant increase was observed in the pivotal il23a and ifng cytokine genes, which was found to be significant after re-examination using the more sensitive qrt-pcr. although no protein data are avaliable, it is tempting to speculate that the combined upregulation of il23a, il and tgfb are indicative of th induction. in fact, although il17a was not represented in the microarray, qrt-pcr findings showed an increase in the mrna levels, suggesting the involvement of th cells in rat tnbs colitis. a contribution of th cells is also likely in view of the ifng upregulation and lack of il changes, although il12a and il12b were not augmented. our group and other authors previously reported an increase in the ifn-γ or ifn-γ/il- protein ratio in this model. a staggering increase was observed in some of the genes, e.g. s100a9/s100a suggesting that they may be highly sensitive markers to follow intestinal inflammation, as previously observed in humans. these genes encode proteins expressed by macrophages and neutrophils and are involved in bacterial defence, chemotaxis and signalling. analysis of the microarray data offered insights into the nature of the inflammatory infiltrate. tnbs colitis was previously characterized at this level. in brief, macrophages and neutrophils are initially recruited to the colonic mucosa and submucosa, followed by a more predominant role of lymphocytes in later stages of colitis, which are characterized by progressive healing, reepithelialization, crypt enlargement and prominent fibrosis. the predominance of neutrophils/macrophages in the inflammatory response was confirmed in our study by the global increase in gene markers of these cell types and the absence of effects on b, t and nk cells. it should be noted that the sample used for genomic analysis did not include lymphoid follicles, in which lymphocytes accumulate in this model, which explains the lack of a significant increase. however, gene ontology analysis indicated that lymphocyte differentiation was already prominent at day our data also revealed important changes that were not directly linked to inflammation/immunity. thus, marked alterations were found in metabolism-related genes, indicating a reduced functionality of numerous biochemical pathways, including glycolysis, purine biosynthesis, krebs cycle, cholesterol biosynthesis and transport, fatty acid biosynthesis and respiratory chain. taken together, these data demonstrate a decrease in biosynthesis of macromolecules, an increase in catabolism, and a decrease in aerobic and anaerobic respiration. therefore, mucosal cells may be have inadequate energy resources during the inflammatory response. similar changes have been reported in ibd patients and in experimental colitis. a number of genes encoding ion transporters were affected by tnbs colitis, including: atp1a encoding the catalytic subunit of the na+/k+ pump; scnn1a, encoding the alpha polypeptide of the epithelial sodium channel; nhe encoding the housekeeping na+/h+ exchanger; and slc12a encoding nkcc the na+/k+/cl- cotransporter. these were generally downregulated, as were the transporters involved in nacl absorption, e.g., nhe nhe and slc26a however, cftr was unchanged. these modifications are consistent with severe alterations of ionic transport, as demonstrated in ibd and animal models. a striking finding of our analysis was the vast number of upregulated genes involved in tissue remodelling in tnbs colitis, generally during the chronic phase, as indicated by the gene ontology analysis. the chronic phase is characterized by mucosal wound healing and submucosal fibrosis and by the scarring and deposition of excess muscular tissue, extending through day and eventually leaving sequelae. these features are often detectable by the naked eye as colonic shortening, deformation and rigidity. two features are related to the intestinal fibrosis suffered by ibd patients: an increase in collagen synthesis by smooth muscle cells, fibroblasts and myofibroblasts, and an increase in muscle layer thickness. igfbp- expression is known to increase in ulcerative colitis, while igf- and tgf-β expression is known to increase in both ulcerative colitis and crohn's disease. these molecules have been related to extracellular matrix remodelling and may therefore be relevant to the fibrosis observed in ibd. our results are in agreement with these ibd findings, showing an increase in igf igfbp and tgfb expression in the inflamed colon. hence, the tnbs model may be appropriate for the study of molecular mechanisms implicated in the development of fibrosis in intestinal inflammation. in addition, expressions of procollagen/collagen genes, most notably tgm and of several enzymes related to collagen processing were increased in the tnbs colitic animals. our results include some intriguing findings, including: the severe repression of nuclear receptors nr1d and nr1d involved in circadian expression patterns and recently shown to be regulated by heme; the synthesis and marked upregulation of tg in the inflamed colon; and the downregulation of the thyroid receptor. further experiments are warranted to clarify the role of these changes in colonic inflammation. we were especially interested in establishing a correlation between the known characteristics of the model and the time course of gene expression. because the time points selected cover all stages of the tnbs-induced inflammatory response, from acute to chronic and healing/recovery phases, we expected to find substantial differences in the pattern of expression. for instance, genes involved in inflammation/immunity and tissue regeneration were expected to predominate in the early and late stages, respectively. however, the vast majority of transcripts followed a common trend, namely a change of expression that was maximal at days and when colitis is most prominent, and was normal at day hence, there is a close correlation between the pathological features of tnbs colitis and the changes observed in the transcriptome. the genes that were up- or downregulated only at days or did not conform to any specific category. some of the transcripts involved are of interest. thus, retnla, which encodes resistin-like alpha, which is dramatically repressed soon after colitis induction, may be involved in monocyte activation, as is the beta isoform. both ace and cma which encode enzymes with angiotensin i cleaving capacity, are induced specifically at day some specific receptors involved in cellular signalling are differentially regulated, such as galr hrh npffr and cnr further analysis is warranted to explore the implications of these findings. despite using a technical approach that meets or surpasses common standards in genomic studies, we carried out more than qrt-pcr determinations in additional samples for a posteriori data validation. as expected, the overall correlation was excellent. in some cases, microarray analysis appeared to be less prone to pick up statistically significant variations, i.e. it was less sensitive than qrt-pcr. the fact that we examined four different time points adds to the complexity of the analysis, since a marked change at one time point may not be enough in many cases to reach significance in the anova. further validation is provided by the fact that the changes observed in many genes, including il1b, il1rn, cftr, tnf, alpl, alpi, ifng, il il pgts nos and ccl among many others, are in line with previous observations from our own group, either at the rna or protein level. previous studies have examined changes in the transcriptome in intestinal inflammation, especially in mouse colitis elicited by tnbs and other manoeuvres. these studies are somewhat limited because they either use small microarrays, examine animals shortly after colitis induction, offer limited or no postgenomic validation or use longitudinal tissue samples. one of these studies addressed the time course of gene expression, but it studied recovery from repeated tnbs challenges. in general, the changes in gene expression documented in the present study are much stronger and wider than those found in previous reports. for example, genes were differentially expressed in the above study in compared to the thousands of genes in our investigation. on the other hand, there have been several human ibd microarray studies, which have detected some key gene expression changes associated with human intestinal inflammation, including some specifically linked to either ulcerative colitis or crohn's disease. however, it is poorly understood how genomic data are related to ibd pathology and therapy, partly because of the variability inherent to human studies and partly because of methodological differences. for instance, the most important source of variation in gene expression measurement is the microarray platform itself, and all of the above studies used different arrays. there are also differences in design and especially in sample selection, i.e., individual vs. pooled or mucosal vs. full thickness. nevertheless, there are some similarities with the rat tnbs model, including increases in chi3l mmp and apoe, s100a ltb, sod cxcl cxcl il and many other genes.  we have characterized the rat tnbs model of ibd at the genomic level, obtaining novel data on individual genes, metabolic pathways and biological functions that are altered during colitis. these findings establish a basis for further mechanistic studies on drug action and research into the pathophysiology of intestinal inflammation. the full genomic database will serve as a reference standard for all future investigations using this model and for comparisons with other animal models of colitis, an approach that is currently being applied in our laboratory. abbreviations ibd: inflammatory bowel disease; il: interleukin; qrt-pcr: quantitative real time pcr; tnbs: trinitrobenzenesulphonic acid. authors' contributions fsmlh and oma conducted the animal experiments, coordinated the bioinformatic analysis of the microarray and qrt-pcr data and drafted the manuscript. fsmlh, oma, mds and az conceived the study. all authors contributed to the interpretation of the data and have read and approved the final manuscript. 
the tnbs model of rat colitis has been described in detail at the transcriptome level. the results were successfully validated in a subset of genes by real-time pcr. we have characterized this model at the genomic level using a longitudinal approach. six control rats were compared with colitic animals at and days after tnbs administration. trinitrobenzenesulphonic acid induced rat colitis is one of the most widely used models of inflammatory bowel disease, a condition whose aetiology and pathophysiology are incompletely understood. most genes were affected at more than one time point. they were related to a number of biological functions, not only inflammation/immunity but also transport, metabolism, signal transduction, tissue remodeling and angiogenesis.
 trinitrobenzenesulphonic acid induced rat colitis is one of the most widely used models of inflammatory bowel disease, a condition whose aetiology and pathophysiology are incompletely understood. we have characterized this model at the genomic level using a longitudinal approach. six control rats were compared with colitic animals at and days after tnbs administration. the affymetrix rat expression array system was used.  tnbs-induced colitis had a profound impact on the gene expression profile, which was maximal and days post-induction. most genes were affected at more than one time point. they were related to a number of biological functions, not only inflammation/immunity but also transport, metabolism, signal transduction, tissue remodeling and angiogenesis. gene changes generally correlated with the severity of colitis. the results were successfully validated in a subset of genes by real-time pcr.  the tnbs model of rat colitis has been described in detail at the transcriptome level. the changes observed correlate with pathophysiological disturbances such as tissue remodelling and alterations in ion transport, which are characteristic of both this model and ibd. 