MDMA users consistently exhibit memory dysfunction but there is an ongoing debate if these deficits are induced mainly by alterations in the prefrontal or mediotemporal cortex, especially the hippocampus. Thus, we investigated the relation of verbal memory deficits with alterations of regional cerebral brain glucose metabolism (rMRGlu) in recreational MDMA users.18F)fluoro-D-glucose positron emission tomography (18FDG PET) in 19 male recreational users of MDMA and 19 male drug-naïve controls. 18FDG PET data were correlated with memory performance assessed with a German version of the Rey Auditory Verbal Learning Test.Brain glucose metabolism in rest was assessed using 2-deoxy-2- of MDMA users. Among MDMA users, learning and recall were positively correlated with rMRGlu predominantly in bilateral frontal and parietal brain regions, while recognition was additionally related to rMRGlu in the right mediotemporal and bihemispheric lateral temporal cortex. Moreover, cumulative lifetime dose of MDMA was negatively correlated with rMRGlu in the left dorsolateral and bilateral orbital and medial PFC, left inferior parietal and right lateral temporal cortex.Verbal learning and recall deficits of recreational MDMA users are correlated with glucose hypometabolism in prefrontal and parietal cortex, while word recognition was additionally correlated with mediotemporal hypometabolism. We conclude that memory deficits of MDMA users arise from combined fronto-parieto-mediotemporal dysfunction. Subsequently, we correlated rMRGlu with several RAVLT parameters in both groups separately to determine the origin of memory dysfunction in MDMA users. The neuropsychological pattern already published from this sample suggested that frontal and mediotemporal dysfunction might be involved in the development of memory deficits Thus, although memory deficits are the most consistent finding in MDMA users Both groups did not significantly differ with respect to age, handedness, years of education, and verbal intellectual performance Table 1.As reported previously from this sample , , MDMA users showed a significant decrease of resting rMRGlu in the right and left DLPFC , Table 4 and To investigate the relation of decreased resting rMRGlu with memory impairment we only investigated positive correlations between rMRGlu and memory performance (low performance in conjunction with decreased rMRGlu) in MDMA users  and Table 5Within the MDMA-group, low rMRGlu in the left inferior parietal (BA 40), left premotor (BA 6), left dorsolateral (BA 8), and left ventrolateral frontal cortex  was significantly correlated with worse performance during the first RAVLT trial (supraspan).Within the MDMA-group, rMRGlu in the right premotor (BA 6), bilateral dorsolateral prefrontal , and the left inferior frontal cortex (BA 47) was significantly correlated with the total learning performance in the RAVLT.Within the MDMA-group, rMRGlu in the bilateral dorsolateral and ventrolateral PFC , the right premotor cortex (BA 6), the right inferior parietal cortex , and the right superior temporal cortex (BA 13), were significantly positively correlated with the delayed recall performance in the RAVLT.Within the MDMA-group, rMRGlu only in the right DLPFC (BA 9) was correlated with recall consistency during the first five RAVLT trials.Within the MDMA-group, rMRGlu in the bilateral superior temporal cortices (BA 38), the right orbitofrontal cortex , the left anterior hippocampus and parahippocampal gyrus (BA 28) and the left parieto-occipital part of the angular gyrus (BA 39) was associated with corrected recognition performance of list A (p(A)) of the RAVLT. and , To investigate the impact of cumulative MDMA intake across lifetime on structural alterations of the brain, we only investigated negative correlations between resting rMRGlu and drug dose (high consumption corresponding with decreased rMRGlu). Low rMRGlu was significantly correlated with high cumulative total dose of MDMA in the left dorsolateral (BA 8) and bilateral orbital and ventromedial PFC  as well as in the left inferior parietal cortex (BA 40) and the right lateral temporal cortex   with the correlation clusters between rMRGlu and delayed recall and between rMRGlu and MDMA intake in the PMOD 3.0 . All three significant cluster sets were projected on an FDG PET template drawn from the control group Figure 4Glucose hypometabolism of MDMA users strongly overlapped with positive correlations of rMRGlu and delayed recall in the bilateral DLPFC . Moreover, there was a triple overlap of MDMA intake, delayed recall, and hypometabolism in the left DLPFC. Finally, drug intake and delayed recall correlations with rMRGlu overlapped in right inferior parietal cortex (BA 40).18FDG-PET in rest, we demonstrated that MDMA users showed a significant reduction of cerebral glucose metabolism predominantly in the bilateral DLPFC and inferior parietal cortex, as well as the bilateral thalamus, right hippocampus, right precuneus, right cerebellum, and pons (at the level of raphe nuclei). In the group of MDMA users, learning and recall parameters were positively correlated with rMRGlu primarily in bilateral frontal and parietal brain regions, while word recognition was additionally related to rMRGlu in the right mediotemporal and bihemispheric lateral temporal cortex. In addition, cumulative lifetime dose of MDMA was negatively correlated with rMRGlu in the left DLPFC, bilateral ventromedial prefrontal and orbitofrontal cortex as well as left inferior parietal and right lateral temporal cortex. Importantly, glucose hypometabolism and rMRGlu correlations with recall deficits and cumulative MDMA intake altogether strongly overlapped within the left DLPFC of MDMA users, indicating that foremost drug-induced changes of the DLPFC might contribute to memory impairment in MDMA users.The aim of this study was to investigate the neurobiological correlates of memory deficits in currently abstinent recreational MDMA users. Using Regarding affected brain structures in MDMA users, we replicated several previous findings reporting decreased glucose metabolism in the DLPFC Previous studies demonstrated that regional reduction of cerebral glucose metabolism might result from loss of neuronal connections and projections Given that we have only investigated current users with a mean duration of abstinence of two to three weeks in a cross-sectional design, it can not be concluded if the reported alterations are lasting or transitory.The results from this study confirm our hypothesis that hypometabolism in the frontal cortex and in the hippocampus is correlated with verbal memory deficits in recreational MDMA users. Within the MDMA group, worse working memory (supraspan) performance was correlated with low rMRGlu inferior parietal, premotor, dorsolateral, and left ventrolateral PFC. Given that the supraspan has a strong working memory component Within the MDMA group, total learning performance was associated with the right premotor cortex, bilateral dorsolateral prefrontal, and the left inferior frontal cortex. Therefore, learning deficits in MDMA users seem to be caused by dysfunction of frontal encoding or recall processes The association of delayed recall and rMRGlu showed the broadest anatomical extension within the MDMA group. Delayed recall performance was positively correlated with the rMRGlu in a prefrontal cluster including the bilateral dorsolateral and ventrolateral PFC and right premotor cortex. Moreover, a parieto-temporal cluster including the right inferior parietal cortex and right superior temporal cortex appeared. Thus, prefrontal and parieto-temporal areas seem to be involved in the dysfunction of delayed recall in MDMA users, indicating disturbed memory formation into–or diminished recall from association cortices, beyond the shown working memory and frontal encoding and recall deficits Corrected recognition performance was associated with rMRGlu in bilateral superior temporal cortices, right orbitofrontal cortex (gyrus rectus), left anterior hippocampus and parahippocampal gyrus, and left parieto-occipital part of the angular gyrus. Therefore, particularly reduced recognition performance in MDMA users might be explained by hippocampal dysfunction Notably, most of the areas showing glucose hypometabolism in MDMA users are part of the two core brain networks: the resting-state default mode network Taken together, the previously postulated dissociation of prefrontal and hippocampal functions as correlates of memory deficits in MDMA users, resulting from temporal or hippocampal dysfunction alone The cumulative total dose of MDMA was negatively correlated with rMRGlu in the left dorsolateral and bilateral orbital and ventromedial PFC, left inferior parietal, and right lateral temporal cortex. This finding is largely in line with a recent study showing significant correlation of the severity of MDMA, heroin, alcohol, and cannabis use with rMRGlu in the DLPFC and lateral temporal cortex of polydrug users A limitation of this study is the use of subjective reports for assessing the extent of drug consumption. The reliability of these reports may be questionable, as they depend on memory function and are probably influenced by anticipated social expectancies. A solution of this problem would be a toxicological hair analysis, which was not available for us at that time. In this regard, our urine drug screenings are only of restricted advantage, as water soluble substances such as MDMA can only be analyzed until few days post exposure 18F-altanserin PET in combination with a dexfenfluramine challenge in order to measure 5-HT release capacity The availability of biological markers for the central serotonergic system such as 5-HT or 5-HIAA CSF or plasma concentrations would have been of advantage for the interpretation of our study results. However, to date such markers have only low validity and reliability 18FDG PET and neuropsychological data show that verbal memory deficits are correlated with glucose hypometabolism in dorsolateral prefrontal and parietal areas of the brains of abstinent MDMA users. This is especially true for global learning performance and delayed recall, while recognition is additionally associated with mediotemporal hypometabolism. Frontal and parietal areas show the highest overlap of glucose hypometabolism with impaired memory function and cumulative drug intake. In conclusion, memory deficits of MDMA users seem to arise from combined fronto-parieto-mediotemporal dysfunction.Our The study was approved by the Ethics Committee of the Medical Faculty of the University of Bonn. After being informed of the aim of the study by written and oral description, all participants gave written informed-consent statements. The study has been conducted according to the principles expressed in the Declaration of Helsinki.Nineteen male, chronic but currently abstinent users of MDMA and 19 subjects with no history of illicit drug use were studied . All participants were recruited by advertisements in a techno music magazine. Subjects of the MDMA group were required to have used MDMA at least 50 times in lifetime and over a period of at least one year. In addition, the use of MDMA clearly had to dominate the consumption of any other psychotropic drug and participants should especially have no substantial previous use of other amphetamine derivatives  or cocaine. PET scans and neuropsychological assessments were carried out when participants were abstinent for at least three days. Inclusion criteria for both groups included negative drug urine toxicology. Exclusion criteria for both groups included use of any psychotropic medication, a present psychiatric disorder, a family history of psychiatric disorders, as well as a severe somatic disease. None of the participants reported a history of migraine, epilepsy, or craniocerebral trauma.Mehrfachwahl-Wortschatz-Intelligenztest was used Structured Clinical Interview (SCID-I) interview according to Diagnostic and Statistical Manual IV (DSM-IV) procedures. Drug history and present pattern of psychotropic drug consumption was assessed by the Interview for Psychotropic Drug ConsumptionOn the first day the neuropsychological test battery was assessed in the afternoon (2 p.m.), while on the subsequent day the PET scan was carried out in the morning (8 a.m.). For the estimation of verbal intellectual performance, the Verbaler Lern- und Merkfähigkeitstestp(A)list A according to p(A) is comparable to the discrimination performance (d-prime) of the signal detection theory Employment of the 18FDG PET scans in rest were conducted according to the 18FDG-European Association of Nuclear Medicine Procedures Guidelines for Brain Imaging using PET (Version I)18FDG as radiotracer. A ten-minute emission scan was started 30 minutes after injection of 296–370 MBq 18FDG. This was followed by a five-minute transmission scan using 68germanium. Image reconstruction was performed due to filtered back projection using a Hannig-Filter (cut-off frequency of 0.5 cycles), thus leading to 47 transversal layers of 128×128 pixels each and voxel sizes of 2.059×2.059×3.375 mm. Blood glucose levels were assessed anterior to the injection of the tracer. Arterialized blood sampling was used to measure 18FDG in plasma. Subjects were scanned once, under resting conditions  during 8:00 and 9:30 a.m., each.On the second day, 2-tests with Yates correction.Age, verbal IQ, years of education, and memory parameters were analyzed using t-tests for independent samples . Handedness and the ratio of smokers and non-smokers were calculated using χMontreal Neurological Institution (MNI), the images were spatially normalized using the PET template provided in SPM5 and subsequently smoothed with an isotropic Gaussian kernel to 12×12×12 mm full width at half maximum (FWHM). The normalized and smoothed images were adjusted by a proportional scaling  for the individual global differences in cerebral glucose metabolism. For voxel-wise comparisons, t-test for independent samples was used. Voxel-wise correlations were performed to assess the relationship between changes in rMRGlu and changes in individual cognitive performance within each group. Because we would have liked to avoid “voodoo correlations” we did not correlate memory performance with metabolic rates within clusters showing significant rMRGlu differences between both groups PET data were analyzed using Statistical Parametric Mapping 5 . According to the convention of the


Embedded in the sequence of each transfer RNA are elements that promote specific interactions with its cognate aminoacyl tRNA-synthetase. Because the anticodon is often a major identity determinant itself, it is possible to switch between certain tRNA functional types by means of anticodon substitutions. By using a synteny-conservation–based method, we detected tRNA anticodon shifts in groups of closely related species and found a total of 75 anticodon shifts: 31 involving switches of identity  and 44 between isoacceptors that code for the same amino acid (isoacceptor shifts). Drosophila, six nematodes, 11 Saccharomycetes, and 61 Enterobacteriaceae. We found a total of 75 anticodon shifts: 31 involving switches of identity  and 44 between isoacceptors that code for the same amino acid (isoacceptor shifts). The relative numbers of shifts in each taxa suggest that tRNA gene redundancy is likely the driving factor, with greater constraint on changes of identity. Sites that frequently covary with alloacceptor shifts are located at the extreme ends of the molecule, in common with most known identity determinants. Isoacceptor shifts are associated with changes in the midsections of the tRNA sequence. However, the mutation patterns of anticodon shifts involving the same identities are often dissimilar, suggesting that alternate sets of mutation may achieve the same functional compensation.Embedded in the sequence of each transfer RNA are elements that promote specific interactions with its cognate aminoacyl tRNA-synthetase. Although many such “identity elements” are known, their detection is difficult since they rely on unique structural signatures and the combinatorial action of multiple elements spread throughout the tRNA molecule. Since the anticodon is often a major identity determinant itself, it is possible to switch between certain tRNA functional types by means of anticodon substitutions. This has been shown to have occurred during the evolution of some genomes; however, the scale and relevance of “anticodon shifts” to the evolution of the tRNA multigene family is unclear. Using a synteny-conservation–based method, we detected tRNA anticodon shifts in groups of closely related species: five primates, 12  Transfer RNAs (tRNA) are involved in two functionally related but physically decoupled reactions that are integral to protein synthesis . First, tRNAs are ∼76 nt in length . The numWhile all aaRSs catalyze the same type of reaction by the same mechanism, they share only a few common structural features. The synthetase family of enzymes can be divided into two distinct classes on the basis of the architecture of the catalytic domain . Class IThe 20 aaRS enzymes must discriminate between a pool of structurally similar tRNAs. Therefore specific elements in tRNA sequence and structure must be present to allow synthetases to recognize and aminoacylate cognate tRNAs and avoid interaction with noncognate tRNAs. However, not all recognition elements (or “identity” elements) are obvious; many tRNAs that accept the same amino acid (isoacceptors) have different nucleotide sequences , and eveThe sequence and structural features that promote recognition by specific aaRSs are termed identity determinants, while those that prevent indiscriminate interactions are termed antideterminants . Known tEscherichia coli tRNA-Gly uses a crucial U73 discriminator base in combination with a C2:G71 pair, while in mammals the discriminator base is A73  gene  gene . A handfGU) gene , in vertGU) gene , in Drososophila , in nemaosophila , and in osophila . One recosophila .Drosophila genus, we have carried out an extensive search for instances of tRNA gene recruitment events. We searched for sets of tRNA orthologs in complete genome sequences of five primates, six nematode worms, 12 Drosophila flies, 11 Saccharomycetes yeast, and 61 Enterobacteriaceae bacteria. We identified instances of tRNA orthologs with different anticodons (anticodon shifts), gene recruitment events involving anticodons of both different tRNA identities , and anticodon mutations that remain associated with the same amino acid (isoacceptor shifts). In order to further study the structural mechanisms of both types of tRNA anticodon shift, we searched for mutations in other regions of the tRNA that covary with the mutations in the anticodon.By using a micro-synteny–based method based on our previous work to identify orthologs of the tRNAs in the Drosophila), six nematode worms (five Caenorhabditis and one Pristionchus), 11 Saccharomycetes yeast (10 Saccharomyces and one Candida), and 61 bacteria from the family Enterobacteriaceae, each one from a different genus . Among the five groups of organisms, there is great variety in the size of the genomes, the number of predicted tRNAs, and the evolutionary divergences involved . After removing low-confidence orthologs with dissimilar sequences and removing tRNAs with unstable structures , we obtained a filtered set of 75 predicted anticodon shifts: 31 alloacceptor shifts (involving a predicted change in tRNA identity) and 44 isoacceptor shifts (a change between anticodons specifying the same identity).P < 0.0001, χ2 test).We considered a typical eukaryotic tRNA complement of 46 tRNA anticodons representing 21 identities (the 20 directly encoded identities and selenocysteine) . Within In total, there are 71 anticodon shifts involving single-base anticodon changes  , and four = 0.88) and with the average genome size (Pearson's r = 0.89). Putative primate anticodon shifts were the most common, comprising 41% of the total detected, despite this group containing only five genomes and the shortest evolutionary divergence of the groups tested. In contrast, no shifts were detected in yeast, and only one shift was detected in Enterobacteriaceae, despite the numerous species in the latter data set (61) and their much higher evolutionary divergences. Despite the high divergence time, lack of orthologous tRNA identifications is not responsible: Our synteny strategy identifies orthologs for 77% of tRNAs in these bacteria. Thus we suggest that the relative absence of anticodon shifts is related to the lower redundancy of tRNAs in these genomes. In Drosophila, nematode worms, Saccharomycetes, and Enterobacteriaceae, we found a greater number of isoacceptor shifts than alloacceptor shifts, at least twice as many in each taxonomic group. However, in primates this trend is reversed; we identified twice as many potential alloacceptor shifts as isoacceptor shifts. The reason behind this excess of alloacceptor shifts in primates is obscure.Among the 30 eukaryotic alloacceptor anticodon shifts, 17 involve a transition between anticodons that couple with different classes of aaRS classes, eight shifts switch between different class I synthetases, and five between class II synthetases . The higP = 0.04, Pearson's correlation).We found isoacceptor shifts involving 14 out of 19 capable tRNA identities . In contrast to the alloacceptor shifts, the isoacceptor shifts show no bias in the synthetase classes in which they occur. Among the 44 isoacceptor switches, we observe 22 in class I synthetase identities and 22 in class II synthetase identities . There iAmong orthologous tRNA families, we find large variation in the number of extra-anticodon mutations and in the positions of these mutations. By using pair-entropy normalized mutual information (MI) as a measure of covariation with the anticodon, we quantified the covariance occurring at each site with respect to the anticodon, across all alignments that have anticodon shifts . The summed MI score across all alignment positions for an anticodon shift is a measure of the number of mutations (including insertions and deletions) at bases other than the anticodon that covary with the anticodon mutation(s). Since prokaryotic aaRS-tRNA systems differ considerably from eukaryotic systems and since we have detected few anticodon shifts in the bacterial data set, the following analyses focus on the eukaryotic data sets only.P = 0.018, ANOVA): Class I synthetase identity switches have an average MI score of 6.3, and switches among class II identities only 2.2. Switches between the two synthetase classes (trans identity shifts) have an average MI of 3.2. The large majority of eukaryotic alloacceptor shifts (25 out of 30) involve a unique pair of identities. The alloacceptor shift identity pairs that occur more than once comprise two Met/Thr shifts and three Lys/Asn shifts.The 30 eukaryotic alloacceptor anticodon switches have a mean summed MI score of 3.9. This varies greatly among the synthetase classes involved (∑MI) that reflects the strength of its covariance with the anticodon over all alloacceptor shifts. The data show that there are substantial differences in covariance at different positions in the consensus tRNA model  are located in the anticodon and acceptor domains; these two distal ends of the structure are directly involved in the key interface functions of the tRNA molecule and are known to be important identity determinants for several aaRS-tRNA systems . HoweverPerhaps the most striking feature of the alloacceptor shift mutations is their heterogeneity. None of the top-scoring positions appears heavily biased toward any particular pair of identities, or any group of species . This is perhaps a surprise and indicates the presence of anticodon-dependent structural constraints in the tRNA molecule separate from those imposed by the aaRSs.There are 11 consensus positions that have a total MI score of >1 SD (1.57) above the mean (2.06), together with a single nonconsensus position between positions 47 and 48, which is associated with multiple isoacceptor shifts . The locThe variable loop has two covarying consensus positions, 44 and 48. Interestingly, in alloacceptor shifts the adjacent position 45 is the predominant site of covarying mutations, while position 44 has none. The only nonconsensus position that is a common mutation site in the isoacceptor shifts is also located in the variable loop. Position 47/48 is mutated in five isoacceptor shifts, only one involving an insertion or deletion and the remaining four being substitutions . The varLike the alloacceptor shifts, the most frequently mutated positions among isoacceptor shifts are all heterogeneous in tRNA identity . They alThe tRNA multigene family comprises very short genes, which can be very highly conserved and yet are subject to high turnover . Using sOur analysis relies on the accurate prediction of tRNA genes and their identities. We use the tool tRNAscan-SE , which hWe have detected tRNA anticodon shifts in all taxonomic groups tested: primates, flies, worms, yeast, and bacteria. We find the largest number of anticodon shifts between five primate species , while only four were detected in the large group of 61 Enterobacteriaceae. The number of shifts detected in each group is correlated with the average size of that group's mapped tRNA gene complement. This suggests that anticodon mutations are facilitated by a high redundancy of tRNA gene in a genome. In species with high tRNA gene copy numbers, loss of one tRNA gene may have relatively little impact on overall tRNA population in the cell for that tRNA functional type.In contrast with the random expectation, we detect more isoacceptor shifts than alloacceptor shifts in all species groups. This implies greater constraint on alloacceptor shifts than isoacceptor shifts, as might be expected. However, we find little difference between the categories in their average numbers of covarying mutations outside the anticodon, as quantified by MI . Mutations that coevolve with alloacceptor anticodon shifts are expected to ensure that the amino acid identity matches the new anticodon. Indeed, many observed mutations do match those sites commonly associated with tRNA identity determinants. However, extra-anticodon mutations associated with isoacceptor shifts are harder to rationalize and have not been previously described. We suggest that these changes may, at least in part, reflect particular properties of mutations to the first (5′) anticodon base, which is the site of nearly all (87%) isoacceptor shift anticodon mutations. Considering that groups of isoacceptor tRNAs can contain different antideterminants encoded by distinct sequence elements  and thatIn recent years, a handful of studies have addressed the functional relevance of tRNA sequence variations in translation, independent of the tRNA's role in aminoacylation. Since both aaRS interaction and ribosomal interaction constrain tRNA structure in a manner dependent on the anticodon, we expect mutations that coevolve with alloacceptor shift mutations to affect both identity elements and ribosomal interaction sites. In contrast, sites that covary with isoacceptor shifts should affect only the ribosomal interaction sites. Indeed, we find some agreement between the anticodon-dependent conservation data of While the top-scoring covarying mutation sites for both alloacceptor and isoacceptor shifts are spread out over the tRNA molecule, there are notable differences between the two classes. Among the most frequently covarying sites in alloacceptor shifts are the tip of the acceptor stem (bases 72 and 73) and the 3′ side of the anticodon loop ; these distal bases of the tRNA structure have been previously described as key identity determinants in a wide range of systems . In isoaThermus thermophilus crystal structure: PDB accession nos. 1GIX and 1GIY)  face the closely adjacent P-site tRNA anticodon (nd 1GIY) . Previound 1GIY) , 2003. IA few covarying sites show preferences for certain synthetase classes and particular base substitutions. However, in general both alloacceptor and isoacceptor shifts show considerable heterogeneity in the extra-anticodon mutations that covary with their anticodon mutations. Many anticodon shifts involving the same transition of identities and anticodon seem to have very different mutations outside the anticodon. We also found at least two instances of extra-anticodon mutations that contradict known identity determinants. However, we note that for some eukaryotic tRNAs , the acceptor stem is known not to contain major identity determinants . In thesAccording to the “ambiguous identity” hypothesis proposed by Drosophila flies, and 61 bacterial species, each from a different genus of the Enterobacteriaceae family. The list of species and genome assembly versions is provided in Supplemental File 1, and we provide GFF lists of our tRNA annotations in Supplemental File 2. We used a flank-mapping method to detect regions of microsynteny conservation, as previously described . The effect of this normalization is to make full dependency between the two sites score 1, regardless of the relative numbers of sequences of each tRNA type present. Normalization by pair entropy has been shown to perform best out of several variations of MI in detection of coevolving positions in protein sequences  greater than zero, we calculated the MI with respect to each of the three anticodon positions; the highest score of the three was taken as the site's MI with respect to the anticodon. The method treats consensus positions and nonconsensus positions uniformly, so substitutions, insertions, and deletions are all potentially detectable as covarying mutations. We summed the scores at each position for all alloacceptor shifts and isoacceptor group shifts, as well as alloacceptor shifts within both synthetase classes and transitions between classes I and II. Structure “heat maps” for each class of shift were drawn using VARNA (For each extra-anticodon alignment position with a site entropy ng VARNA .Supplemental material is available for this article.


Vascular complications are the leading cause of mortality and morbidity in patients with diabetes. However, proper animal models of diabetic vasculopathy that recapitulate the accelerated progression of vascular lesions in human are unavailable. In the present study, we developed a zebrafish model of diabetic vascular complications and the methodology for quantifying vascular lesion formation real-time in the living diabetic zebrafish.fli1:EGFP, lyz:EGFP, gata1:dsRed, double transgenic zebrafish of gata1:dsRed/fli1:EGFP were exposed to high cholesterol diet and 3% glucose (HCD-HG) for 10 days. The zebrafish model with HCD-HG treatment was characterized by significantly increased tissue levels of insulin, glucagon, glucose, total triglyceride and cholesterol. Confocal microscopic analysis further revealed that the diabetic larvae developed clearly thickened endothelial layers, distinct perivascular lipid depositions, substantial accumulations of inflammatory cells in the injured vasculature, and a decreased velocity of blood flow. Moreover, the vascular abnormalities were improved by the treatment of pioglitazone and metformin.Wild type zebrafish (AB) and transgenic zebrafish lines of A combination of high cholesterol diet and high glucose exposure induces a rapid onset of vascular complications in zebrafish similar to the early atherosclerotic vascular injuries in mammalian diabetic models, suggesting that zebrafish may be used as a novel animal model for diabetic vasculopathy. The latest statistics of National Institute of Health (NIH) indicate that diabetes is the seventh leading cause of death in the United States; vascular complications are the principal cause of morbidity and mortality in patients with type 1 and type 2 diabetes.Zebrafish are widely used to investigate developmental biology due to the advantages of relatively low cost, rapid lifecycle, and the optical transparency of zebrafish larvae. Similar to mammals, the zebrafish pancreas is comprised of two types of glandular tissues, both of which carry out essential physiological functions. While there is no suitable animal models that could recapitulate human diabetic vasculopathy available, A high cholesterol diet induced vascular lipid accumulation and inflammation in zebrafish larvae have also been proposed as a model for real-time study of early atherogenesis in vivo. In the present study, we developed a combination of high cholesterol diet and high glucose environment to induce vascular abnormalities in zebrafish larvae that are characterized with metabolic disturbances similar to type 2 diabetes. We also established methodologies for the quantification of some metabolic parameters and the tracking of vascular abnormalities in living larvae.Zebrafish maintenance and procedures were conducted in accordance with National Institutes of Health guidelines of the use and care of experimental animals and approved by the Institute Animal User and Ethical Committees at Shandong University. High cholesterol diet (HCD) was made by soaking egg yolk in a diethyl ether solution of cholesterol (Solarbio) to achieve a content of 10% (w/w) cholesterol in the food after ether evaporation. To determine the location and amount of vascular lipid accumulation in larvae, both control and HCD food were supplemented with 10 µg/g of a fluorescent cholesteryl ester analog, cholesteryl BODIPY®558/568 dodecanoic acid (Invitrogen). Detailed method can be found in Thirty larvae of each experimental group were euthanized, and washed twice in double distilled water. Abdomens containing undigested food were removed, and the remaining body parts were pooled and homogenized as described previously.Ten larvae were euthanized and homogenized as mentioned above. Thereafter, the tissues were used for isolation of total RNA using TRIzol (Invitrogen) according to the manufacturer's instructions. The isolated RNA was reverse transcribed (Fermantas) and cDNA was stored −20°C. For fluorescent detection of PCR products, reactions containing template and specific primers were amplified with sybergreen .For in vivo confocal microscopy, anaesthetized zebrafish larvae were fixed in a cell culture dish (NEST) with glass bottom in low melt point agrose (Solarbio) as described previously.Data were expressed as means ±SD and values were compared using Student's t test with two-tailed p<0.05 being considered statistically significant.Considering hyperglycemia and hyperlipidemia frequently co-exist in human type 2 diabetes,In our pilot studies, we applied wild type zebrafish larvae with different concentrations of glucose (1%∼5%) and high cholesterol diet . Using a fluorescent cholesteryl BODIPY®558/568 dodecanoic acid, which enables microscopic tracking in vivo, we explored the optimal glucose concentrations and feeding time periods that could result in lipid accumulation in the vasculature of larvae as analyzed by real-time confocal microscopy. We showed that it took more than 3 weeks to induce lipid accumulation in the vascular wall with glucose concentrations of 1% or 2%, whereas glucose concentrations of 4% or 5% caused a high mortality (>75%) within 10 days (data not shown). At a concentration of 3% glucose, a high cholesterol diet induced a clear lipid deposit in the vasculature within 10 days and ∼90% of the larvae survived for more than 15 days during the exposure.To determine whether the formation of vascular lesion is linked to the onset of diabetes under this experimental condition, we examined metabolic profile of wild type zebrafish larvae in the control group and the HCD-HG treatment group. Because of the small size of a larva, it is not feasible to directly collect blood samples. Therefore, we measured total cholesterol and triglyceride levels in the whole body lysate as described previously.In addition, since there are no special antibodies or other reagents that target the insulin and glucagon in zebrafish larvae, we examined the effect of HCD-HG on the corresponding gene expression levels by real-time quantitative PCR. Due to the small body size of the zebrafish larvae, it is practically not possible to isolate specific tissues for the tissue specific mRNA quantifications without noticeable contaminations from surrounding tissues. Therefore, we examined the effect of HCD-HG on the corresponding gene expression levels by real-time quantitative PCR using the mRNAs extracted from the whole body lysate. As the expression level of phosphoenolpyruvate carboxykinase (PEPCK), a critical enzyme that catalyzes the conversion of oxaloacetate to phosphoenol pyruvate, is negatively correlated with the level of blood glucose in acute hyperglycemia while up-regulated by chronic hyperglycemia in type 2 diabetes, We then studied whether the HCD-HG treatment-induced diabetes is associated with the development of vascular abnormalities in zebrafish larvae. We applied real-time confocal microscopic analysis of endothelial cell damage, vascular lipid accumulation and inflammatory cell infiltration, and blood flow velocity in transgenic zebrafish larvae in which endothelial cells, myeloid cells, and red blood cells are genetically labeled with fluorescent proteins. Moreover, we tested the therapeutic efficacy of thiazolidinedione (TZD) pioglitazone and biguanide metformin, well-established anti-diabetic drugs, fli1:EGFP larvae  and at the same time immersing in the 3% glucose solution for 10 days resulted in the appearance of diffused orange fluorescence in the vasculature, consistent with circulating fluorescent lipid. Furthermore, the HCD-HG treatment led to accumulation of bright orange fluorescence in focal areas of blood vessels including the dorsal longitudinal anastomotic vessel (DLAV), caudal aorta (CA) and the caudal vein (CV) (Using ein (CV) , suggestlyz:EGFP larvae, in which EGFP is constitutively expressed in granulotytes/microphages, we tracked the recruitment of GFP+ myeloid cells to the caudal veins within 2-cell distance from the lumen for each experimental group. While only occasional GFP+ cells was observed in the vasculature of larvae fed control diet, there was a robust increase in GFP+ cell recruitment in the HCD-HG fed larvae  in the caudal artery for each experimental group. In the control larvae fed with normal diet, we observed that red blood cells moved with different speeds in the caudal arteries: the highest speed of 1576 µm/s, the lowest of 161 µm/s, the majority distributed between 300∼1100 µm/s with an average of 678±242 µm/s, which is similar to those previously reported.Using double transgenic Despite numerous hypotheses, mechanisms of diabetic vasculopathy are still not fully understood. It has been widely accepted that prolonged exposure to hyperglycemia, dyslipidemia, inflammation, and coagulopathy could contribute to diabetic micro- and macro-vascular diseases. However, large scale clinical trials have revealed that lowering glucose levels does not always translate to the reduction in cardiovascular events in patients with type 2 diabetes. To develop the zebrafish model that has metabolic disturbances similar to type 2 diabetes, we treated the zebrafish larvae with high cholesterol diet and exposed them to high glucose solution, which caused lipid and glucose metabolic disorder in vivo. The treated larvae displayed several vascular changes that are similar to vascular changes in mammalian diabetic models and human subjects with diabetes. This was accompanied by significantly increased glucose, insulin and glucagon levels and the related gene expressions, all of which indicated possible insulin resistance. The treated zebrafish larvae also showed high level of total cholesterol, which is consistent with the observed vascular pathogenesis in HCD-HG treated zebrafish. Lipid accumulation in vessel wall and surrounding tissues suggest that an early atherosclerotic injury might have occurred. We also found thickening of endothelial layer in both optical vessels and caudal vein; inflammatory myeloid cells infiltrated into the vascular walls; and reduced blood flow. These early vascular changes were attenuated when pioglitazone or metformin were administered at the same time of the HCD-HG treatment. Both drugs partly inhibited the endothelial thickening, inflammatory activation and lipid accumulation in the caudal vein. However, pioglitazone and metformin did not restore the decreased blood flow caused by the HCD-HG.Like most other animal diabetic models, the current zebrafish model also has limitations. As a lower vertebrate, the pathophysiological conditions of zebrafish differ from those in humans and mammals. The observed vascular changes may also not be manifested with the same pathological presentations as those in larger mammals and human subjects. Not all features in vascular changes as observed in other animal models, such as mice or rabbits, or in human subjects can be attributed to the vascular changes we observed in diabetic zebrafish. Furthermore, zebrafish larvae are too small to acquire accurate biochemical estimations individually as we routinely do in large animals. Their whole bodies needed to be pooled in order to measure these biomarkers. Since there were no antibodies specifically against the zebrafish insulin and glucagon, levels of these proteins could not be quantified in this study.In the meantime, the zebrafish possess many unique advantages, such as low cost, short lifecycle and large scale drug screening capacity. More specifically, zebrafish can be easily genetically manipulated and tested in vivo longitudinally. Based on these points, the zebrafish diabetic model may be best utilized as a large scale drug screening tool in addition to its applications to the mechanistic research of diabetic vascular complications. However, findings from this model may require further validation in large animal models.In summary, we believe that the zebrafish model established in this study manifests several biochemical features similar to type 2 diabetes, and is an informative experimental model for investigations of early stage diabetic vascular complications, which is a crucial time window for effective therapeutic interventions. Complemented with availability of large number of genetically manipulated zebrafish, this model can be used to evaluate the role of specific molecular pathways in vascular diseases and therapeutic agents that may specifically regulate the pathways. It can serve as the first line animal model to screen agents that may be potentially developed to treat diabetic cardiovascular diseases.Supporting Information S1Details of the experimental methods are provided in the supporting information.(DOC)Click here for additional data file.


Removal of visual cortex in the rat axotomizes projection neurons in the dorsal lateral geniculate nucleus (dLGN), leading to cytological and structural changes and apoptosis. Biotinylated dextran amine was injected into the visual cortex to label dLGN projection neurons retrogradely prior to removing the cortex in order to quantify the changes in the dendritic morphology of these neurons that precede cell death. At 12 hours after axotomy we observed a loss of appendages and the formation of varicosities in the dendrites of projection neurons. During the next 7 days, the total number of dendrites and the cross-sectional areas of the dendritic arbors of projection neurons declined to about 40% and 20% of normal, respectively. The response of dLGN projection neurons to axotomy was asynchronous, but the sequence of structural changes in individual neurons was similar; namely, disruption of dendrites began within hours followed by cell soma atrophy and nuclear condensation that commenced after the loss of secondary dendrites had occurred. However, a single administration of fibroblast growth factor-2 (FGF2), which mitigates injury-induced neuronal cell death in the dLGN when given at the time of axotomy, markedly reduced the dendritic degeneration of projection neurons. At 3 and 7 days after axotomy the number of surviving dendrites of dLGN projection neurons in FGF-2 treated rats was approximately 50% greater than in untreated rats, and the cross-sectional areas of dendritic arbors were approximately 60% and 50% larger. Caspase-3 activity in axotomized dLGN projection neurons was determined by immunostaining for fractin (fractin-IR), an actin cleavage product produced exclusively by activated caspase-3. Fractin-IR was seen in some dLGN projection neurons at 36 hours survival, and it increased slightly by 3 days. A marked increase in reactivity was seen by 7 days, with the entire dLGN filled with dense fractin-IR in neuronal cell somas and dendrites. Dendritic alterations, such as the formation of focal swellings, the appearance of varicosities, and the loss of distal segments, are found in many neurons in the CNS following various injuries When the brain is injured traumatically, neurons that project to the site of injury commonly are disconnected from their synaptic targets. This disconnection, or axotomy, typically initiates a cascade of molecular and cellular events leading, in many cases, to the death of the disconnected cells in vivo model for studying the response of neurons in the central nervous system to axonal injury. Projection neurons in the rat dLGN send their axons to the visual cortex, and, therefore, these neurons can be axotomized by removing the visual cortex The projection neurons in the dorsal lateral geniculate nucleus (dLGN) of the adult rat offer an excellent At 3 days after axotomy, cytological and biochemical changes are seen in the cell somas of many axotomized neurons; e.g., atrophy, nuclear condensation and DNA fragmentation, and the upregulation of caspase-3 activity The earliest structural changes are observed in the dendrites of axotomized dLGN projection neurons at 12 hours after a lesion of visual cortex. In contrast, cell somas of axotomized projection neurons do not display obvious atrophy until 3 days after a cortical lesion, at which time many axotomized dLGN projection neurons have lost over 50% of their dendrites. During the next four days, massive cell death in the dLGN ensues, with many injured dLGN projection neurons displaying cytoplasmic vacuoles, disrupted membranes and nuclear condensation.Many trophic factors, such as nerve growth factor (NGF), fibroblast growth factor-2 (FGF2), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and glial cell-derived neurotrophic factor (GDNF), have been demonstrated to mitigate the severity of neuronal loss after injury or disease FGF2, a member of a family of proteins that bind heparin and heparan sulfate, is involved in a large number of biological activities and plays a crucial role in the maintenance, survival and selective vulnerability of various neuronal populations in the normal, injured, or diseased brain Cell soma atrophy, the condensation of nuclear chromatin, and subsequent DNA fragmentation are generally believed to indicate that an injured neuron is undergoing apoptotic cell death We investigated caspase-3 activity in axotomized dLGN projection neurons immediately following injury using an antibody raised against fractin, a 32 kDa N-terminal actin fragment that results exclusively from the cleavage of beta actin by activated caspase-3 All animal handling and procedures were performed in accordance with protocols for these studies that have been approved by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison. All surgery was performed aseptically under deep anesthesia, and every attempt was made to minimize pain and discomfort.Sixty-three adult male Holtzman rats (250–275 grams) were used in this study. Forty-eight rats received bilateral injections of biotinylated dextran amine (BDA) into the visual cortex and five rats received injections of Texas Red conjugated dextran (TRD) into the right visual cortex. Three days after making the BDA or TRD injections, all of the injected rats, as well as 10 additional, uninjected animals, received a unilateral lesion of the right visual cortex. Twelve animals that received both bilateral injections of BDA and a lesion of the visual cortex also received a single administration of FGF2 into the lesion cavity (see below). The rats then were allowed to survive for periods ranging from 6 hours to 7 days before being deeply anesthetized and perfused through the heart with 4% paraformaldehyde.Animals were anesthetized initially with an intramuscular injection of a mixture of 70 mg/kg of ketamine HCl and 7 mg/kg of xylazine. After being placed in a stereotaxic head holder, anesthesia was maintained with 1–2% isoflurane. Under aseptic conditions, bone flaps were cut bilaterally to expose the dorsal surface of the visual cortex, cortical areas 17, 18, and 18a  as definAnimals were anaesthetized as described and placed in a stereotaxic head holder. Under aseptic conditions, a bone flap was cut over the dorsal surface of the right visual cortex to expose cortical areas 17, 18, and 18a . Five 0.Three days after the injection of BDA or TRD, the animals were anesthetized again. The wound clips placed previously were detached, and the bone flap above the right visual cortex was removed to re-expose the visual cortex, cortical areas 17, 18 and 18a , which wThe FGF2 treated rats and untreated rats were allowed to survive for 6, 12, 24, 48, 72 hours, or 7 days after the lesion of visual cortex (n = 6 for each group), and then they were deeply anesthetized with a mixture of 135 mg/kg of ketamine HCl and 14 mg/kg of xylazine and perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were post-fixed overnight in 4% paraformaldehyde and then sectioned coronally at 75 µm with a vibrating blade microtome . Sections through the dLGN were collected and washed thoroughly in 0.1 M phosphate buffer. Retrogradely transported BDA was detected by using an avidin-biotin-peroxidase complex  and visualized using diaminobenzidine (DAB) as a chromogen. Finally, the sections were processed for gold/silver intensification and prepared for examination under brightfield microscopy Fifteen rats were used for the evaluation of caspase-3 activity. The animals survived for 24, 36, 48, 72 hours or 7 days after the visual cortex had been removed (n = 3 each group), and then were anesthetized, perfused with 4% paraformaldehyde, and their brains were sectioned at 40 µm as described above. Caspase-3 activity was detected by using a rabbit polyclonal antibody (a gift from Dr. Greg Cole of UCLA) that had been raised against the C-terminus of a 32-kDa N-terminal actin fragment, fractin, which is generated specifically by caspase-3 cleavage t-test. Differences between means were considered significant if they exceeded p<0.01.All BDA-labeled neurons were examined with a brightfield microscope at a magnification of 1000X. Since the BDA labeling quality varied significantly among individual dLGN projection neurons, structural analysis was restricted to projection neurons that appeared to be completely labeled. Using the same criteria as described previously t-test. Differences between means were considered significant if p<0.01.At 36, 48 and 72 hour survival times, it was possible to distinguish individual fractin-IR neurons in the dLGN ipsilateral to a lesion of the visual cortex. At each of these survival times the number of fractin-IR neurons in the dLGN was counted. The group means ± the standard error of the mean were calculated, and differences between group means were evaluated statistically with an unpaired Student's two tailed The structure of dLGN projection neurons retrogradely labeled with BDA in normal rats has been described in detail in a previous report Twelve hours after axotomy, cytological changes of various degrees were evident in some BDA-labeled projection neurons, although some axotomized neurons, principally radial cells, were morphologically similar to control neurons with normal-sized cell somas that supported many secondary dendrites decorated with appendages. While many radial cells displayed relatively minor structural changes at 12 hours survival, basket cells  often exWithin the next 36 hours, the number of BDA-labeled projection neurons with degenerated dendrites increased steadily, while the number of labeled projection neurons with nearly normal dendrites gradually disappeared. The majority of BDA-labeled neurons lost many of their dendritic branches and distal segments during this period, but their cell somas, primary and some secondary dendrites appeared relatively normal. However, labeled projection neurons with multiple long dendrites were occasionally seen , but the2, and none of the remaining dendrites was longer than 150 µm. In these projection neurons, some primary dendrites remained, but only short remnants of secondary dendrites were evident .Quantitative analysis showed that axotomized dLGN projection neurons in FGF2-treated rats had, on average, 50% more dendrites than untreated controls at 72 hours and 7 days survival. Similarly, the dendritic arbors of axotomized dLGN projection neurons in FGF2-treated rats were 60% larger than those seen in axotomized cells in untreated rats at 72 hours survival and 50% larger at 7 days survival . StatistFractin-IR was seen only in regions of the brain with predictable cell death based upon a region's relation to the damaged visual cortex. For example, fractin-IR was observed in the ipsilateral cortex near the site of the lesion, and in a few of the dorsal-lateral thalamic nuclei that are known to project to the visual cortex. However, our analysis of the development of fractin-IR focused on neurons in the dLGN ipsilateral to the visual cortex lesion. It is important to note that rats studied for fractin-IR with a biotinylated secondary antibody and DAB did not receive cortical injections of BDA to avoid confounding BDA-labeling with fractin labeling. Nevertheless, it is reasonable to conclude that the observed fractin-labeled neurons in these rats are projection neurons as less than 1% of dLGN interneurons die or undergo apoptosis following a visual cortex lesion In rats that survived for 24 hours after a lesion of the visual cortex, fractin-IR was not seen in the dLGN ipsilateral to the cortical lesion, but at 36 hours survival, scattered fractin-IR in the ipsilateral dLGN was evident. This fractin-IR appeared mainly as loose clusters of fractin-positive puncta that appeared to be scattered along the dendrites of dLGN projection neurons . OccasioThe number of fractin-IR neurons increased slightly within the next 12 hours, resulting in the detection of 2.9 fractin-IR cells, on average, in each dLGN section. Although the number of fractin-IR neurons in the dLGN was greater at 48 hours survival than at 36 hours survival, the distribution and appearance of the fractin-IR neurons was similar at the two survival periods . HoweverDuring the next four days, fractin-IR in the dLGN increased dramatically, and at 7 days survival dense fractin-IR puncta filled the entire dLGN ipsilateral to the cortical lesion. Because of the intense overlap of fractin-IR cells and processes, it was difficult at 7 days survival to distinguish individual fractin-IR neurons. Nevertheless, it was possible to discern that many dLGN neurons displayed fractin-IR in their nuclei  and someTo confirm that axotomized dLGN projection neurons were fractin positive, we identified projection neurons that had been labeled retrogradely following injections of TRD into the visual cortex. FITC immunofluorescence was used to detect fractin-IR in sections through the dLGN. Neurons that had been labeled with TRD (red) and were positive for fractin (green) were recognized as fractin-IR dLGN projection neurons .These results demonstrate the involvement of activated caspase-3 in the death of axotomized projection neurons in the dLGN, and show that caspase-3 activity is evident as early as 36 hours after axotomy, and that it develops slowly and asynchronously in injured dLGN neurons during the next 12 hours. The expression of activated caspase-3 then increases dramatically in the following 24 hours and is observed in all of the cellular compartments of dLGN neurons that have been affected by a lesion of the ipsilateral visual cortex.In this study, we have used BDA as a retrograde tracer to demonstrate the structural changes that occur in the dendrites of confirmed dLGN projection neurons following axotomy during very early and later stages of cell death. In addition to confirming that cytological changes that take place in the cell somas of axotomized projection neurons in the dLGN at relatively late stages of injury, 3 days and thereafter, we demonstrated a series of structural changes in the dendrites of injured dLGN neurons as early as 12 hours after axotomy, well before changes in cell somas can be observed. This report also provides evidence that axotomized dLGN projection neurons in the rat can survive for at least 7 days after axotomy. Nevertheless, none of the observed surviving projection neurons appeared normal, which is perhaps to be expected as approximately 70% of the neurons in the dLGN ipsilateral to a cortical lesion have died at 7 days after a lesion of the visual cortex In order to rescue injured neurons from death and to reduce injury-induced functional deficits, it is necessary to understand the cytological and molecular changes that occur sequentially in neurons following an injury, particularly during the period before injured neurons commit to dying. Axotomized projection neurons in the dLGN proceed through an orderly series of events before dying, perhaps leaving open a “window of opportunity” during which time the death of some injured neurons may be averted by the administration of appropriate pharmacological agents or neuroprotective factors.While injured neurons usually display various cytological changes, the best characterized changes have focused on cell soma atrophy, nuclear condensation and DNA fragmentation; all relatively late stage occurrences when cell death has become an irreversible fate choice of an injured neuron. By contrast, relatively little attention has been paid to the structural changes that occur in neurons soon after an injury is experienced. In adult rats, counts of neuronal cell somas show that approximately 99% of dLGN neurons ipsilateral to a lesion of the visual cortex remain intact during the first 72 hours after the lesion is made, but during the next four days 66% of the neurons in the dLGN die in vivo insults Dendritic changes have been reported by others in CNS neurons after various This result raises the possibility that neurons that have experienced an injury that results in structural changes in their dendrites may retain the potential to survive if the early, injury-induced dendritic degeneration can be blocked from proceeding further. Initial changes in dendritic morphology resulting from injury, such as the formation of varicosities or beading, may not have a significant effect on cell survival, while a loss of secondary dendrites is likely to lead to cell death as secondary or higher-order dendrites support the majority of dendritic appendages that are critical for inter-neuronal communication. Therefore, when an injured neuron has lost its secondary or higher-order dendrites, it also loses its ability to communicate with other neurons, which results in neuronal dysfunction and often cell death. In this study, we observed that only those axotomized projection neurons that retained secondary dendrites, even if relatively few in number and short in length with respect to their normal counterparts, survived for at least seven days after axotomy. This observation suggests that maintaining a minimum number of secondary dendrites after axotomy may be a necessary requirement to extend neuronal survival and prevent rapid cell death.As described here, dLGN projection neurons displayed an orderly sequence of dendritic alterations during the first 72 hours after axotomy before cytological changes in their cell somas were seen. As early as 12 hours after axotomy, the dendrites of injured dLGN projection neurons had begun to lose their appendages and distal segments of secondary dendrites. Within the next 48 hours, axotomized projection neurons gradually lost all of the distal segments of their dendrites. The sequence of dendritic degeneration, from the loss of appendages and the formation of varicosities to the disappearance of secondary dendrites, generally was consistent among cells in each of the three classes of projection neurons that we have described and from cell to cell within each class.However, the initiation, duration and endpoint of the sequence varied among axotomized dLGN projection neurons. Thus while all dLGN projection neurons in this study experienced a comparable injury and displayed similar structural alterations in their dendrites soon after axotomy, the extent of these alterations varied with the class of projection neuron, and often differed among neurons within each class of projection neurons, regardless of the survival time after axotomy. Among the three classes of projection neurons, cells that retained four or more primary dendrites after axotomy were radial or basket cells. In contrast, bipolar cells experienced severe dendritic degeneration and disappeared from the dLGN earlier than basket or radial cells. These results suggest that the three classes of dLGN projection neurons differ in their vulnerability to axotomy, bipolar cells being more vulnerable than basket cells which in turn are more vulnerable than radial cells. The reasons for this differential vulnerability remain to be determined, but it may be related to the number of afferent synapses that each class of projection neuron can support after axotomy. The number of dendrites, and, therefore, the opportunity for synaptic connections is greater in radial cells than in basket cells and least in bipolar cells In summary, dendritic degeneration is a very early event in adult rat dLGN neurons after axotomy. Changes in the dendritic structure of dLGN projection neurons emerge within 12 hours after axotomy. These dendritic changes continue during the next 48 hours, during which time most dLGN projection neurons lose their secondary dendrites. The disappearance of secondary dendrites is accompanied by cell soma atrophy, which is underway 72 hours after axotomy. Since dLGN projection neurons undergo a predictable sequence of structural alterations after axotomy, these alterations may be used as criteria to discriminate among different degenerative stages of injured neurons, and may be useful as reference points to investigate the biochemical and molecular events that underlie cell death after axotomy at defined stages.In this report we also have confirmed caspase-3 activity in axotomized dLGN projection neurons using an antibody raised against an actin fragment, fractin, which results solely from the cleavage of beta actin by activated caspase-3. While calpain may cleave actin in addition to caspase-3, fractin is a specific 32 kD product produced solely by the cleavage of actin by caspase 3 as reported by several previous studies that involved: (a) the immunolabeling of Hirano bodies, which are eosinophilic rod-shaped inclusions found primarily in CA1 hippocampal neurons in aged animals and in the brains of Alzheimer's disease patients However, it is important to note that the caspase-3 activity that has been detected in these experiments may underestimate its activity, because beta actin is only one of several substrates of caspase-3. Moreover, caspase-3 may be activated earlier than reported here to cleave other substrates, such as α-spectrin and gelsolin, which have not been examined in this study. Nevertheless, the present results demonstrate clearly that caspase-3 is active in axotomized dLGN neurons, and they illustrate the temporal and spatial pattern of this activity in projection neurons in the dLGN ipsilateral to a lesion of the visual cortex.Axotomized dLGN neurons have been thought to die apoptotically, because they display morphological characteristics typically associated with apoptosis That caspase-3 also may be activated later than other proteases in axotomized dLGN projection neurons is consistent with the observation in the present study that the appearance of intense fractin-IR in the dLGN occurs first at 72 hours after axotomy, well after dendritic degeneration is initially detected, and is followed soon thereafter by a period of massive cell death in the dLGN Activated caspase-3 also is implicated in the death of axotomized dLGN neurons in young rats and mice. In rats, removal of the visual cortex during the first two postnatal weeks results in the rapid, massive death of neurons in the dLGN with complete loss of the nucleus by adulthood In contrast, in axotomized dLGN projection neurons in the adult rat fractin-IR was detected first in dendrites, the most vulnerable compartment of these neurons at 36 hours survival. Only after the intensity of fractin-IR had increased in both dendrites and cell somas at 72 hours survival was fractin-IR detected in cell nuclei, which often appeared condensed. It is perhaps not a coincidence that the dendrites of axotomized dLGN projection neurons degenerate progressively during the first three days after axotomy. At 72 hours survival, approximately 60% of the dendrites of axotomized dLGN projection neurons have been lost, and cell somas have begun to atrophy and display nuclear condensation. These results indicate that the temporal progression of caspase-3 activity in axotomized dLGN projection neurons follows with a delay the same sequence of structural changes that are observed in these neurons; specifically, the structural changes seen in axotomized dLGN projection neurons and caspase-3 activity appear first in the dendrites of the injured neurons, and then in the next two days advance to involve the cell soma, preceding the death of the injured neuron. In an axotomized dLGN projection neuron, the progression of cytoskeletal degeneration and caspase-3 activity from dendrites to the cell soma is a roadmap leading to the death of the injured neuron. Interrupting this roadmap may offer an opportunity to protect axotomized neurons from dying.With this in mind, we investigated interrupting the roadmap with a single administration of FGF2 at the site of the cortical lesion to determine qualitatively and quantitatively if this affected the time course and extent of the dendritic degeneration in axotomized projection neurons in the rat dLGN. The present results clearly demonstrate that a single administration of FGF2 significantly reduces, but does not prevent, the degeneration of dendrites in these axotomized neurons.As mentioned, when the dendrites of injured dLGN projection neurons degenerate, the ability of these neurons to receive information from other cells and participate functionally in neuronal circuits is compromised. When axotomized projection neurons in the dLGN of the rat lose more than 50% of their dendrites, they frequently die. By contrast, when dLGN projection neurons retain more than 10 dendrites and a dendritic arbor with a cross-sectional area at least 20% of normal size, some projection neurons survive up to 7 days after axotomy. Thus, maintaining a minimum number of dendrites and a dendritic arbor of minimal dimensions appears to be associated with the survival of dLGN projection neurons after axotomy. Therefore, a reduction in dendritic degeneration following axotomy may play an important role in protecting injured dLGN projection neurons from dying.in vivo has been shown to reduce neuronal death in the adult brain triggered by axotomy Administration of FGF2 Of interest is that the effect of FGF2 treatment on reducing the loss of dendrites in axotomized dLGN projection neurons was observed for all three classes of projection neurons. In normal rats bipolar cells are the most vulnerable dLGN projection neurons to axotomy, nearly disappearing as a cell class of projection neurons at 7 days. By contrast, radial cells are the most resistant projection neurons, and many of them survive in the dLGN 7 days after axotomy. However, in FGF2-treated rats, all three classes of dLGN projection neurons are seen 7 days after axotomy, and, notably, some bipolar cells with normal appearing dendritic trees are present at 7 days .It is important to note, however, that the protection afforded by a single treatment with FGF2 does not affect all axotomized dLGN projection neurons uniformly. While neurons from each of the three classes of dLGN projection neurons, radial, basket and bipolar cells, often appeared nearly normal in FGF2 treated rats 7 days after axotomy , other rL expression while decreasing the expression of the pro-apoptotic protein Bax, and blocked the cleavage of caspase 3 L, the down regulation of pro-death proteins, such as Bax, PARP or DFF, and the blockage of caspase-3 cleavage.Although the neuroprotective effects of FGF2 after axotomy have been widely acknowledged, as mentioned, the intracellular signaling mechanisms involved remain unclear. In cultured rat cortical neurons, FGF2 treatment increased ERK1/2 phosphorylation resulting in prolonged neuronal survival In summary, the degenerative changes in the dendrites of dLGN projection neurons can be observed as early as 12 hours after axotomy, and are among the most rapidly occurring consequences of cell injury. That FGF2 retards the degeneration of the dendritic cytoskeleton and increases the survival of dLGN projection neurons after axotomy strongly suggests that FGF2 may inhibit proteases such as calpain and caspase-3 from cleaving the cytoskeletal proteins that are essential to dendritic integrity, as well as interfering with the cleavage of pro-apoptotic proteins that are directly involved in initiating apoptotic cell death. Since cytoskeletal protein degradation is the direct cause of the axotomy-induced changes in cell structure that ultimately may lead to cell death, it is reasonable to suggest that the efficacy of FGF2 as a neuroprotective factor is due in part because it may function as an inhibitor of cytoskeletal or pro-apoptotic proteases upon binding to an injured neuron.


Even though obesity is a well-established risk factor of type 2 diabetes, there is emerging evidence that persistent organic pollutants (POPs), a variety of lipophilic chemicals accumulated in adipose tissue, may be critically involved in the pathogenesis of type 2 diabetes. Among various POPs, serum concentrations of organochlorine pesticides and polychlorinated biphenyls (PCBs) were the most strongly and consistently linked to type 2 diabetes in both cross-sectional and prospective studies. In particular, obesity did not seem to be related to type 2 diabetes among persons with very low serum concentrations of POPs, suggesting a more fundamental role of chlorinated POPs in the pathogenesis of type 2 diabetes. These POPs were also associated with obesity, insulin resistance, and adverse lipid profiles like high triglyceride and low high-density lipoprotein (HDL) cholesterol among persons without diabetes, all metabolic dysfunctions commonly observed before developing type 2 diabetes. Recent animal studies supported the findings from epidemiological studies. If all these findings on POPs are true, it suggests that any effort to reduce the external and internal exposure to POPs would be necessary to decrease the social burden of type 2 diabetes. Type 2 diabetes is one of the most common chronic diseases in both developed and developing countries and continues to increase in numbers and significance as economic development and urbanization lead to changing lifestyles. Type 2 diabetes is usually associated with obesity, physical inactivity, older age, or family history of diabetes. Among known risk factors, obesity is the most important and well-established one. However, there is emerging evidence that persistent organic pollutants (POPs) accumulated in adipose tissue, rather than obesity itself, may be critically involved in the pathogenesis of type 2 diabetes.POPs are a group of chemicals with common properties such as lipophilicity and persistence. The examples of POPs are polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, polychlorinated biphenyls (PCBs), and organochlorine (OC) pesticides. Although most chlorinated POPs such as PCBs and OC pesticides were banned several decades ago and dioxins are strictly regulated in most developed countries, the general population is currently exposed to these chemicals through food consumption because they have widely contaminated our food chain, especially fatty animal food. In addition, POPs which were accumulated in adipose tissue from previous high exposure are a source of internal exposure as they keep being release from adipose tissue to circulation and reach critical organs.In a cross-sectional study among the U.S. general population published in 2006, serum concentrations of chlorinated POPs were strongly associated with the prevalence of type 2 diabetes . After aHowever, these findings seemed to be puzzling because the body burden of chlorinated POPs has declined worldwide over the recent several decades after banning of production and usage in most developed countries while type 2 diabetes show a recent worldwide epidemic. If chlorinated POPs are really important in the development of type 2 diabetes, how is this kind of mismatch of time trend possible? Our explanation on this issue was that a low dose but persistent exposure might be more harmful than high dose exposures if chlorinated POPs are involved in the pathogenesis of type 2 diabetes as endocrine disruptors.Larger effects from low dose exposure than high dose exposure have been proposed as possible biological responses of chemicals with endocrine disrupting properties . It is dThese cross-sectional associations were recently confirmed by several prospective studies, even though the kinds of individual POPs predicting type 2 diabetes and the shapes of the dose-response curves were not exactly the same among the studies -10. AlsoImportantly, the prospective studies among young adults showed clear inverted U-shaped associations ,9. In thAnother interesting finding was that some POPs even predicted future obesity . ConsistIn conclusion, the results from a series of recent studies strongly suggest that environmental exposure to some chlorinated POPs may be involved in the increased risk of type 2 diabetes, as well as obesity and other obesity-related metabolic disturbances. If all these findings on POPs are true, it means that any effort to reduce the external and internal exposure to POPs would be necessary to decrease the social burden of type 2 diabetes.


Critical illness polyneuropathy (CIP) is a severe complication of critical illness. The clinical features of CIP are muscle weakness and atrophy causing delayed weaning and prolongation of the mobilization phase. Although the exact etiopathogenesis has not yet been fully elucidated, sepsis, systemic inflammatory response syndrome, and multiple organ failure seem to play an important role. CIP is diagnosed by signs of denervation in electromyography. Although there is no causal treatment for CIP, retrospective data suggest that early IgM-enriched intravenous immunoglobulin (IVIG) application may prevent or mitigate CIP. Therefore we aimed to investigate the use of IVIG in the early treatment of CIP in critically ill patients in a prospective, randomized, double-blind and placebo-controlled setting.In this prospective, randomized, double-blind and placebo-controlled trial critically ill patients with clinical evidence for incipient CIP, a diagnosis of SIRS/sepsis and failure of at least two organ systems were randomized to be treated either with IgM-enriched IVIG or with human albumin 1% as placebo over a period of 3 days. The primary objective was to demonstrate that administration of IVIG prevents and/or mitigates CIP in critically ill patients, measured by electrophysiological stimulation of the median, ulnar and tibial nerves on days 0, 4, 7 and 14. Electrophysiological measures were graded according to compound muscle action amplitude size (CIP score) of the respective nerve. Secondary objectives were mortality from any cause within a 28-day period and lengths of ICU stay.n = 19) or placebo (n = 19). Baseline characteristics including CIP score on day 0 were similar between the two groups. CIP could not be improved significantly by IVIG treatment for three consecutive days, represented by similar CIP scores of all three measured nerves on days 4, 7 and 14 in the IVIG and the placebo group. Mean CIP score levels of all three nerves significantly increased from baseline to day 4 in both groups.Thirty-eight critically ill patients were included and randomized to either receiving IgM-enriched IVIG (Results suggest that early treatment with IVIG neither significantly improves CIP nor influences the length of stay or mortality in critically ill patients. Consistent with the literature, CIP deteriorated during the course of disease in critically ill patients with a diagnosis of SIRS/sepsis and failure of two organ systems.


Motor neuron diseases (MNDs) are characterized by selective death of motor neurons and include mainly adult-onset amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Neurodegeneration is not the single pathogenic event occurring during disease progression. There are multiple lines of evidence for the existence of defects in lipid metabolism at peripheral level. For instance, hypermetabolism is well characterized in ALS, and dyslipidemia correlates with better prognosis in patients. Lipid metabolism plays also a role in other MNDs. In SMA, misuse of lipids as energetic nutrients is described in patients and in related animal models. The composition of structural lipids in the central nervous system is modified, with repercussion on membrane fluidity and on cell signaling mediated by bioactive lipids. Here, we review the main epidemiologic and mechanistic findings that link alterations of lipid metabolism and motor neuron degeneration, and we discuss the rationale of targeting these modifications for therapeutic management of MNDs. SOD-1), TAR DNA binding protein of 43-kDa (TDP-43), fused in sarcoma (FUS) and chromosome 9 open reading frame 72 (C9ORF72)  are a group of incurable neurological disorders caused by the selective degeneration of motor neurons. Amyotrophic lateral sclerosis (ALS) is the most representative MNDs among adults with a incidence rate of 2–3 per 100,000 -1 alpha  is a key enzyme for the regulation of fatty acid metabolism, and it can impact fatty acid oxidation taking place in mitochondria. SCD-1 introduce a double bond in the carbon chain of saturated, to generated mono-unsaturated fatty acids that are more prone to be stored in fat tissues. We have recently reported a downregulation of SCD-1 in the muscle of SOD1 mice , directly determines membrane fluidity and movement of membrane proteins in lipid rafts  is rare subtype of ALS characterized by loss of lower motor neurons. To our knowledge, only one report described alterations of circulating lipids in two patients with a PMA syndrome  is downregulated in the muscles of SBMA mice. PLA2g7 degrades phospholipids to release poly-unsaturated fatty acid and, in muscle, it is involved in the differentiation of myoblasts , already used in multiple sclerosis to lower neuroinflammation in the CNS. In addition to pharmacological agents, dietary supplementation with neuroprotective and anti-inflammatory fatty acids, or derivatives, is worthy of further characterization. Altogether, alterations of lipid metabolism in MND deserve further work that will undoubtedly lead to new and interesting therapeutic options for patients.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our review will highlight a specific role for calcium as a signaling messenger in activating focal clusters of integrins bound to the cytoskeleton which allows the cell to attain a migratory phenotype. The precise interplay between chemokines, selectins, and integrins binding under the ubiquitous presence of shear stress from blood flow provides an essential cooperative signaling mechanism for effective leukocyte recruitment.Leukocyte trafficking to acute sites of injury or infection requires spatial and temporal cues that fine tune precise sites of firm adhesion and guide migration to endothelial junctions where they undergo diapedesis to sites of insult. Many detailed studies on the location and gradient of chemokines such as IL-8 and other CXCR ligands reveal that their recognition shortly after selectin-mediated capture and rolling exerts acute effects on integrin activation and subsequent binding to their ligands on the endothelium, which directs firm adhesion, adhesion strengthening, and downstream migration. In this process, G-protein coupled receptor (GPCR) signaling has been found to play an integral role in activating and mobilizing intracellular stores of calcium, GTPases such as Rap-1 and Rho and cytokeletal proteins such as Talin and F-actin to facilitate cell polarity and directional pseudopod formation. A critical question remaining is how intracellular Ca In the circulation, β2-integrins are expressed on the membrane at low numbers and in a low affinity state that rapidly shift to high affinity and increase in number, and surface density as they make contact with endothelium at sites of inflammation. Affinity is regulated via allosteric changes in integrin structure that in turn modulate their adhesion potential. Following selectin dependent capture and rolling, an upshift occurs from a low affinity bent conformation to an extended conformation associated with intermediate affinity that can bind to endothelial ligands and effect deceleration of rolling leukocytes. Chemokines play a key role in signaling a shift in integrin conformation from intermediate to high affinity that is associated with adhesive stabilization, such that the leukocyte becomes resistant to tensile and shear repulsive forces of blood flow. In fact, it is control of the number and density of high affinity integrins and endothelial presentation of their cognate ligands that determines when and where leukocytes are recruited to emigrate during inflammation  and Diacylglycerol (DAG) that triggers an elevation in intracellular calcium level through release of PLC dependent ER stores , which then mobilizes Inositol-1,4,5 triphosphate  is a versatile signaling molecule that is critical to synchronizing rolling, arrest and polarization events during leukocyte migration. Ca2+ transients are spatially and temporally regulated by communication between the calcium stores in the endoplasmic reticulum (ER) and membrane distributed calcium channels activated through GPCR signaling and integrin engagement with their ligands on the endothelium. We have mentioned above how chemokine activation through GPCRs is followed by an intracellular Ca2+ burst mediated through PLCs that is necessary to trigger integrin activation and leukocyte arrest. This Ca2+ flux serves to activate downstream messengers that include calpain, calmodulin, GTPases, and Talin1, some of which also regulate superoxide production, and exocytosis of secretory granules containing additional integrins and proteolytic enzymes  that is mediated by both transient receptor potential (TRP) channels and Orai1, 2, and, 3 that control store operated calcium entry  activation (Di Paolo et al., 2+ influx through CRAC and cooperation with GPCR release of ER stores for initial Kindlin-3 association is yet to be elucidated. Our laboratory has been examining the role of Kindlin-3 in PMN arrest and adhesion strengthening in shear flow. We observe that Kindin-3 binding to the β-subunit of LFA-1 is critical for adhesion strengthening of arrested PMN at high shear stress and facilitates rapid clustering of LFA-1 at focal sites that engage ICAM-1. This data thus supports previous studies highlighting Kindlin-3 as a key player in mediating adhesion strengthening through β1 integrins and its dispensable role in GPCR mediated integrin affinity upshift (Hyduk et al., 2+ via Orai1 CRAC that cooperates with ER stores to effect a local burst in Ca2+ concentration (Figure There is much recent interest in the roles of Talin1 and Kindlin-3 as key cytoskeletal adaptor proteins in the regulation of integrin affinity and clustering during the transition from neutrophil rolling to arrest and shape polarization as it navigates to sites of transmigration on inflamed endothelium (Sarantos et al., n Figure  Figure 22+ mediated signaling of adhesion stabilization and integrin outside-in signaling (Marks and Maxfield, 2+ are critical to facilitating F-actin polymerization and integrin polarity during migration (Schaff et al., 2+ flux. Blocking SOCE with CRAC channel inhibitors, using siRNA, or genetic deletion to knockdown Orai1 expression all result in impaired neutrophil arrest, polarization, and abrogation of directional migration under shear flow (Schaff et al., 2+ entry through Orai1 and STIM1 drives focal adhesion turnover through Ras and Rac1 and together play a vital role in tumor metastasis (Yang et al., Remarkable is the high frequency of immunodeficiency diseases that are associated with mutations in the effector molecules that directly influence affinity modulation and clustering of integrins. These include Orai1, Kindlin-3, WASp, CalDAG-GEF1, and Vav1, all of which have been identified in leukocyte adhesion deficiencies. Moreover, all of these components cooperate with Ca2 integrin structure, mutations in the fucosyl transporter gene required for producing sialyl-Lewisx selectin ligands that support leukocyte rolling on the endothelium, and a general defect in integrin activation of β1, β2, and β3 integrins, respectively (Abram and Lowell, Immunodeficiencies have also been linked to impaired GTPases, GEFs, and cytoskeletal protein signaling. Leukocyte adhesion deficiency I, II, and III occur due to defects in β2-integrins and severely impaired adhesion and migration of neutrophils on inflamed endothelium (Zhang et al., Similar to Kindlin-3, WASp also connects the actin cytoskeleton to integrin cytodomains to facilitate leukocyte migration via control of integrin adhesion functions. A crucial effector of Rho GTPases and an important activator of the Arp2/3 cytoskeletal complex, WASp deficiency leads to Wiskott-Aldrich syndrome that is characterized by increased susceptibility to infections (Thrasher, 2+ converges with influx through CRAC, thereby providing a means to dynamically modulate the number and location of integrin bonds and subsequent migration. This is accomplished by shear stress mediated tensile force transmission, which requires bond formation at sites in which β2-integrins are engaged at sufficient bond strength and density where their survival is ensured. At these locations, high affinity LFA-1 associates with cytosolic Kindlin-3 thus enabling association of a complex with Orai1 that together transduce a local increase in Ca2+. This in turn activates membrane diffusion of additional high affinity LFA-1 to bond with available endothelial ICAM-1. Further, local cytosolic release of Ca2+ promotes the assembly of cytoskeletal elements including Talin and F-actin to the integrin tail in a complex that provides the machinery for adopting a polarized elongated shape as a neutrophil extends pseudopods and initiate transendothelial migration. In this manner, mechano-transduction through integrins provides a means for sensing the direction and magnitude of shear force via a complex that involves at a minimum LFA-1, Orai1, Kindlin-3, Talin1, Vav-1, and WASp. These molecules enable neutrophils to efficiently navigate the journey from the blood stream to inflammatory sites that is critical for host defense.With each heartbeat, leukocytes make a fateful decision when they encounter vascular sites of inflamed endothelium; to arrest or not to arrest. This singular event multiplied by millions of encounters can determine the intensity of the neutrophilic response to infectious or autoimmune tissue insults. Assisting in this decision process is the relative density of chemokines and selectins expressed on inflamed endothelium that facilitate neutrophil activation by ligating their respective cognate receptors on the tethered cell. In this review, we detailed how cytosolic release of CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Under common practice, recognition and treatment of type 2 diabetic nephropathy (DN) are usually revealed at a rather late stage (CKD stages 3–5) due to the insensitiveness of available diagnostic markers. Accumulating data obtained from vascular homeostasis in late stage DN demonstrated (1) a defective angiogenesis and impaired NO production which explains the therapeutic resistance to vasodilators and the inability to correct chronic renal ischemia and (2) an abnormally elevated antiangiogenesis and a progressive vascular disease which correlates with the altered renal hemodynamics characterized by a progressive reduction in renal perfusion as the disease severity progressed. In contract, the vascular homeostasis is adequately functional in early stage DN. Thus, vasodilator treatment at early stage DN (CKD stages 1-2) can enhance renal perfusion, correct the renal ischemia, and restore renal function. Similarly, microalbuminuria is usually detected when the creatinine clearance is approaching fifty percent level as depicted in Under current practice, determinations of serum creatinine and microalbuminuria are used to screen for diabetic nephropathy (DN), which is reflected by microalbuminuria  and serum creatinine concentration greater than 1 mg/dL. In accordance with the 5 stages of chronic kidney disease (CKD), serum creatinine usually does not change at the early stage  With respect to VEGF, we have studied VEGF value in the serum, which partly reflects a component of circulating endothelial cell-receptor-bound VEGF detached from the diseased vascular wall into the circulation. Our study demonstrated a wide range of VEGF values with the mean value of VEGF not significantly different from the control. This observation of VEGF in diabetic patient is quite contrast to the reduced VEGF value observed in the nondiabetic chronic kidney disease patient . Further(II) With respect to defective VEGFR1 documented in this study in late stage of DN, the activation of VEGF through the classical VEGF → VEGFR 1 pathway would be defective, impairs physiologic Akt phosphorylation, uncoupling of eNOS, and thus impairs NO production. Previous study confirmed this view in vitro when VEGFR 1 was knocked down in endothelial cells using retroviral small interfering RNA for VEGFR 1, but not VEGFR 2; the cGMP was lower in VEGFR 1 knocked down endothelial cells, suggesting a decreased NO bioavailability . (III) With respect to the defective endothelial progenitor cell and angiopoietin 1, diabetic patients have been reported to have a reduced number of circulating endothelial progenitor cell as well as its function, with the extent of reduction directly proportional to plasma hemoglobin A1C level , 25. A d(IV) With respect to the abnormally elevated antiangiogenic VEGFR2 observed in late stage of DN, other studies showed conflicting results. Cooper et al.  demonstrπB activation, then activates ERK-p38, JAK-STAT, and therefore induces VSMC proliferation. Moreover, the increase in transmural pressure associated with intraglomerular hypertension would, in accordance with La Place's law, translate into an increase in biaxial tensile stress which then transmit into the wall of arteriole and enhance further the VSMC proliferation in the vascular wall. The VSMC proliferation would induce a thickening of vascular wall, a narrowing of vascular lumen, and eventually a progressive reduction in peritubular capillary flow. Collectively, these would incriminate in the pathologic development of neoangiogenesis, a progressive macro- microvascular disease. In this regard, Osterby and Nyberg [In accordance with the increased VEGFR2 observed in late stage of DN, it would pathologically activate Akt phosphorylation through the NO-independent pathway to induce endothelial cell proliferation and endothelial cell dysfunction. Previous study demonstrated that this excessive Akt activation under an impaired NO production would instigate the angiogenic response to VEGF, negatively regulat endothelial cell lifespan and inhibiting endothelial tubular formation via a p53/p21-dependent pathway . In the d Nyberg  described Nyberg . Min andd Nyberg , 40.(V) With respect to altered vascular homeostasis and angiogenesis in diabetic retinopathy, expressions of VEGFA, VEGFR1, and VEGFR2 were all increased in the retina of diabetic or insulin-resistant rats . Such chIn contrast to the abnormal vascular homeostasis and angiogenesis observed in late stage of DN, our study demonstrated the values of vascular homeostasis observed in early stage of DN  not significantly different from the control population . This imIn conclusion, the preceding information renders support that implementation of treatment at the early stage of DN under the environment favorable for renal angiogenesis and regeneration would effectively restore renal function and prevent the end-stage renal disease.