Stroke is the major cause of death and disability worldwide, and the thrombolytic therapy currently available was unsatisfactory. 14-3-3ε is a well characterized member of 14-3-3 family, and has been reported to protect neurons against apoptosis in cerebral ischemia. However, it cannot transverse blood brain barrier (BBB) due to its large size. A protein transduction domain (PTD) of HIV TAT protein, is capable of delivering a large variety of proteins into the brain. In this study, we generated a fusion protein TAT-14-3-3ε, and evaluated its potential neuroprotective effect in rat focal ischemia/reperfusion (I/R) model. Western blot analysis validated the efficient transduction of TAT-14-3-3ε fusion protein into brain via a route of intravenous injection. TAT-14-3-3ε pre-treatment 2 h before ischemia significantly reduced cerebral infarction volume and improved neurologic score, while post-treatment 2 h after ischemia was less effective. Importantly, pre- or post-ischemic treatment with TAT-14-3-3ε significantly increased the number of surviving neurons as determined by Nissl staining, and attenuated I/R-induced neuronal apoptosis as showed by the decrease in apoptotic cell numbers and the inhibition of caspase-3 activity. Moreover, the introduction of 14-3-3ε into brain by TAT-mediated delivering reduced the formation of autophagosome, attenuated LC3B-II upregulation and reversed p62 downregulation induced by ischemic injury. Such inhibition of autophagy was reversed by treatment with an autophagy inducer rapamycin (RAP), which also attenuated the neuroprotective effect of TAT-14-3-3ε. Conversely, autophagy inhibitor 3-methyladenine (3-MA) inhibited I/R-induced the increase in autophagic activity, and attenuated I/R-induced brain infarct. These results suggest that TAT-14-3-3ε can be efficiently transduced into brain and exert significantly protective effect against brain ischemic injury through inhibiting neuronal apoptosis and autophagic activation. Stroke is responsible for more than 5 million deaths each year worldwide, making it the second leading cause of death and a major cause of disability Apoptosis has been suggested to be one of the main contributors to neuronal death in acute ischemic stroke 14-3-3ε is a member of the 14-3-3 protein family, which bind to proteins and modulate interaction between proteins involved in various cellular functions such as intracellular signaling, cell cycling, apoptosis and transcriptional regulation However, 14-3-3ε is unable to cross the BBB due to its large molecular weight of 30-kDa Autophagy, a cellular lysosome-mediated process, degrades and recycles subcellular organelles Whether 14-3-3ε can be delivered into brain by TAT and TAT-14-3-3ε fusion protein has a protective effect against cerebral I/R injury is not known. In the present study, we prepared a TAT-14-3-3ε fusion protein, and evaluated its ability to cross BBB and transduce into the brain. We further examined the neuroprotective effect of TAT-14-3-3ε against rat transient focal cerebral ischemia. We demonstrated for the first time that systemic delivery of TAT-14-3-3ε protein results in a significant neuroprotection, indicating that TAT-14-3-3ε could be developed as a potential therapeutic agent in brain ischemic injury.The experimental designs and all procedures were in accordance with the guidelines for the Care and Use of Laboratory Animals approved by Beijing Committee on Animal Care and Use. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Peking University Health Science Center (Permit Number: LA2013-69). Efforts were made to reduce the number of animals used and their suffering.14-3-3ε gene (GenBank accession number NR_024058) was generated by PCR. In order to facilitate the protein expression in E. coli, we modified the least preferred codons into the most preferred ones without changing amino acid sequence, according to the codon usage in E. colipEASY-T1 cloning vector  by TA cloning and confirmed by DNA sequencing.The sequence encoding human NdeI and 3′ end BamHI sites. The PCR product was subcloned into the NdeI-BamHI digested pET-30a(+) plasmid  resulting in recombinant expression vector pET-TAT-14-3-3ε. In addition, another plasmid pET-14-3-3ε coding for a 14-3-3ε control protein was constructed in a similar way. The insert in all the constructs was verified by DNA sequencing.The 855 bp coding sequence of TAT-14-3-3ε fusion protein, which contains a 6×histidine tag leading sequence, followed by 11-amino-acid TAT-PTD and 14-3-3ε, was obtained by PCR. An oligonucleotide coding for a flexible spacer Gly-Gly-Ser was inserted to flank TAT 5′ and 3′, respectively. Restriction sites were added into the PCR primers to give the PCR products containing 5′ end E. coli BL21(DE3). A single clone was grown for 8 h at 37°C in 10 ml LB medium containing 50 μg/ml kanamycin. These cultures were diluted 50-fold with fresh LB media and cultured at 37°C to OD600 = 0.8. The expression of TAT-14-3-3ε and 14-3-3ε were induced by addition of 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) for 10 h at 28°C. The bacterial cells were harvested by centrifugation at 10,000×g, and then resuspended in pre-cooled binding buffer  and lysed by sonication on ice.The plasmid pET-TAT-14-3-3ε/pET-14-3-3ε was transformed into the 2+-NTA resin column (Invitrogen) pre-equilibrated with binding buffer. The proteins were then eluted by a stepwise imidazole gradient in elution buffers . Eluted fractions were analyzed by 12% (wt/vol) SDS/PAGE, followed by Coomassie blue staining. Fractions that contained target protein were then pooled and dialyzed against water using a 5 kDa molecular weight cutoff dialysis membrane  at 4°C thoroughly. The purified proteins were lyophilized and kept at −80°C. The proteins were dissolved in normal saline (NS) and the protein concentrations were estimated by BCA protein assay  before used, and the protein purity was also evaluated by SDS/PAGE. The purified recombinant proteins were confirmed by Western blot using anti-His-tag antibody  and anti-14-3-3ε antibody .The supernatant containing target protein was collected by centrifugation 15,000×g for 20 min at 4°C and loaded onto a NiFocal cerebral ischemia was induced by transient occlusion of the middle cerebral artery (MCAO) using the intraluminal filament technique as described previously Adult male Sprague–Dawley rats  were given free access to food and water. Rats were randomly assigned to five groups: sham operated group, vehicle group (NS treated), 14-3-3ε treated group, and TAT-14-3-3ε treated group before or after ischemia . Animals were sacrificed at 24 h of reperfusion after 2 h transient cerebral ischemia. Both 14-3-3ε and TAT-14-3-3ε were dissolved in NS. Intravenous injection of 0.3 ml NS (vehicle group), or 10 mg/kg 14-3-3ε (14-3-3ε treated group), or 10 mg/kg TAT-14-3-3ε (TAT-14-3-3ε pre-ischemic treated group) was performed 2 h before MCAO. In group V (post-ischemic treated group), 10 mg/kg TAT-14-3-3ε was intravenously injected at the end of ischemia.In addition, the transduction of TAT-fusion protein across BBB often exhibits a significant increase at 4 h after administration To determine whether the protective effects of TAT-14-3-3ε against transient focal cerebral ischemia is related to autophagy inhibition, rats were randomly assigned to the sham, vehicle, 3-MA and RAP + TAT-14-3-3ε groups. The treatment protocols and dosages of 3-MA and rapamycin were selected according to published studies 24H40O4·Na, 0.1% SDS, 2 mM Na4P2O7, 25 mM β-glycerophosphate, 1 mM Na3VO4 and 1 mM PMSF) 4 h after intravenous injection of TAT-14-3-3ε or 14-3-3ε. Equal amounts of protein (40 μg) were diluted in 6×sample buffer, boiled and separated by 12% (wt/vol) SDS/PAGE, and then blotted onto PVDF membrane  using a wet transfer system . The membrane was then immersed in blocking solution [5% milk in TBST (0.1% Tween 20 + TBS)] for 2 h at room temperature, followed incubated with a monoclonal rabbit anti-14-3-3ε antibody , rabbit anti-His-tag antibody  or rabbit anti-β-actin antibody  overnight at 4°C. Subsequently, membranes were incubated with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody , followed by ECL detection . The density of each band was quantified using the Quantity One software.Transduction of the TAT-14-3-3ε fusion protein was verified using Western blot analysis. Proteins were extracted from the cerebral hemisphere in cold RIPA buffer  After scoring neurological function, the animals were anesthetized with chloral hydrate  and transcardially perfused with NS. Brains were collected rapidly and sectioned into five 2-mm-thick coronal slices, and stained with 1% 2,3,5-triphenyltetrazoliumchloride (TTC)  at 37°C for 30 minutes. The slices were fixed in 4% formalin overnight and then photographed with a digital camera . Unstained areas  were defined as ischemic lesions. The areas of the infarction were analyzed using Image J software (NIH Image), by measuring the surface of each slice and numerically integrated across the thickness of the slice to obtain the infarct volume. Volumes from all five slices were summed to calculate total infarct volume over the entire hemisphere, expressed as mmin situ apoptosis detection kit . Brains were cut into 10 μm thick coronal sections at the level of the bregma, and then incubated with a TUNEL mixture containing 0.5 U/μl TDT, 0.2 nmol biotin-11-dUTP, followed by streptavidin-FITC. TUNEL staining was detected under a fluorescence microscope (Olympus IX71). Three sections from each animal were viewed by two observers blinded to the origin of the slides. For each section, the TUNEL positive cells were counted in 10 non-overlapping high-power fields (×200) covering the majority of striatum.Rats were anesthetized deeply with chloral hydrate  and transcardially perfused with 4% paraformaldehyde (n = 3). Then brains were removed and fixed in 10% buffered formalin solution for 48 h, and then embedded in paraffin. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was performed to investigate the apoptosis according to the protocol of 2, 2 mM EDTA, 0.1% Triton 100, 2 mM dithiothreitol (DTT) and 1 mM PMSF. The homogenates were centrifuged at 13,000 ×g for 30 min at 4°C, and the supernatants were collected. The protein concentration was determined by BCA assay  and adjusted to 2 μg/μl. Then 100 μg proteins were added to 50 μl 2×Reaction Buffer supplement with 5 μl Caspase-3 Substrate and then incubated at 37°C away from light for 4 h. Caspases-3 activities were quantified spectrophotometrically at a wave length of 405 nm in a microplate reader . The activity of caspase-3 was expressed as the fold increase over the sham group.The activity of caspase-3 was detected using a Caspase-3 Colorimetric Assay Kit  following the manufacturer's instruction. Proteins were extracted from fresh right cortical samples 24 h after MCAO (n = 4). Tissues were homogenized in cold lysis buffer containing 25 mM HEPES, pH 7.5, 5 mM MgClTo evaluate the degree of ischemic neuronal damage, Nissl staining was performed according to classical methods. The brain sections described above were stained with cresyl violet . Cells that contained Nissl substance were considered to be viable neurons. Three sections from each animal (n = 4) were selected for analysis. The viable neurons were quantified by manually counting cells with visible nucleoli in 10 randomly chosen, non-overlapping high power fields (×200) of striatum using microscopy (Olympus IX71) in a blinded fashion.For autophagy analysis, rats were subjected to 2 h MCAO followed by 24 h reperfusion, and then proteins were extracted from the ischemic cerebral hemisphere and detected by Western blot analysis. Polyclonal rabbit anti-LC3B antibody , polyclonal rabbit anti-Beclin-1 antibody  and polyclonal rabbit anti-p62/SQSTSM1 antibody  were used for immunoreactivity detection.TEM was used to evaluate the ultrastructural changes. After 24 h of reperfusion, rats were deeply anesthetized and sacrificed by transcardial perfusion with 50 ml NS followed by 50 ml of fixative . Next, the cerebral fragments were fixed with 2.5% glutaraldehyde solution overnight at 4 °C, followed by immersion in 1% osmium tetroxide in 0.1 mol/l phosphate buffer (pH 7.4) for 2 h. The tissues were dehydrated in ascending series of ethanol before embedding samples in araldite. The ultrathin sections were cut and doubly stained with uranyl acetate and lead citrate, and examined with a JEM-1400 type transmission electron microscope  and photographed.All values are expressed as means ± SD. The one way ANOVA was used to test for differences among groups, followed by post hoc Scheffe's tests. P<0.05 was considered statistically significant.Codon-optimized 14-3-3ε DNA sequence was generated by a two-step PCR strategy as shown in Western blot analysis using anti-14-3-3ε antibody showed that the expression levels of endogenous 14-3-3ε protein in brain tissues of I/R rats were decreased by half , confirmTo determine whether the increased 14-3-3ε protein levels in the brain can protect neurons against ischemic damage, we examined infarct volume and neurological deficits after treatment with recombinant 14-3-3ε proteins in rat focal cerebral I/R model. We first detected the function of TAT-14-3-3ε at three doses  . A signiTo explore the cellular mechanisms underlying the neuroprotective efficacy of TAT-14-3-3ε, we examined the apoptotic death by using TUNEL staining and caspase-3 activity colorimetric assay. The number of apoptotic cells in the striatum zone was significantly reduced in both pre-treatment and post-ischemic treatment groups with TAT-14-3-3ε, as compared with vehicle or control 14-3-3ε protein treated group . The casTo further delineate the neuroprotective mechanisms of TAT-14-3-3ε in ischemic brain damage, we examined the effect of TAT-14-3-3ε on I/R-induced autophagy by TEM analysis and Western blot analysis with autophagy specific antibodies against microtubule-associated protein 1A light chain 3 (LC3), autophage-related gene 6 (Atg-6)/Beclin-1 and p62/SQSTM1. Ultrastructural analysis  with TEMWe further tested whether the regulation of TAT-14-3-3ε on autophagy contributed to the neuroprotective effect of TAT-14-3-3ε in ischemic stroke. First, we determined the effect of autophagy on ischemic injury in a rat model using autophagy inhibitor 3-MA. Rats were subjected to 2 h MCAO followed by 24 h reperfusion. The result showed that i.c.v. injection of 600 nmol 3-MA at the onset of reperfusion reduce the infarct size. In contrast, combined treatment (2 h before MCAO) of TAT-14-3-3ε and autophagy inducer rapamycin  partly attenuated the protective effects of TAT-14-3-3ε on infarct volume  and 3. Win vivoThe salient finding in this study is the significant neuroprotection of TAT-14-3-3ε against focal cerebral ischemia. We prepared a novel TAT-14-3-3ε fusion protein using codon-optimization and recombinant protein expression/purification technology. TAT-14-3-3ε readily crossed BBB and effectively transduced brain following intravenous injection even under intact BBB conditions. Pre- or post-ischemic treatment with TAT-14-3-3ε significantly reduced cerebral infarct size and improved neurologic outcomes in a rat focal cerebral I/R model. Such neuroprotection is attributed to the improved neuronal survival due to the significant reduction of neuronal apoptosis mediated by caspase-3 activity, and the inhibition of autophagy. These data confirm the neuroprotective effect of 14-3-3ε via inhibiting neuronal apoptosis in vitro, such as TAT-Bcl-2△loop in vivo. Besides, there were still many TAT fusion proteins showing effect in animal model, but the cellular distribution of these proteins was not yet analyzed, such as TAT-GDNF TAT-mediated transduction is able to cargo large molecules such as proteins across cell membranes, and is becoming a promising tool for the therapy of many diseases Consistent with previous reports Accumulating evidences suggest that 14-3-3ε displays important anti-apoptotic characteristics Autophagy has a beneficial role or adverse effect in cerebral ischemia, depending on various pathological conditions In summary, TAT-14-3-3ε was prepared and transduced into the brain efficiently, and it exhibited significant protective effect against cerebral I/R injury in rats. The mechanisms underlying the neuroprotection of TAT-14-3-3ε in brain ischemia may be attributed to its inhibition on neuronal apoptosis and autophagy. TAT-14-3-3ε might be a new pharmaceutical target for the treatment of not only stroke but potentially other human injuries and diseases.Table S1Primers for synthesis of 14-3-3ε gene.(DOCX)Click here for additional data file.


The present study was designed to investigate the expression profile of α1D-AR in the diabetic kidneys and its modulation by activation of peroxisome proliferator-activated receptors (PPARs). 12-week-old Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were treated with fenofibrate or rosiglitazone for 8–10 weeks. Gene microarray, real-time PCR, and confocal immunofluorescence microscopy were performed to assess mRNA and protein expression of α1D-AR in rat kidney tissue. Using microarray, we found that α1D-AR gene was dramatically upregulated in 22-week-old ZD rats compared to ZL controls. Quantitative PCR analysis verified a 16-fold increase in α1D-AR mRNA in renal cortex from ZD animals compared to normal controls. Chronic treatment with fenofibrate or rosiglitazone reduced renal cortical α1D-AR gene. Immunofluorescence staining confirmed that α1D-AR protein was induced in the glomeruli and tubules of diabetic rats. Moreover, dual immunostaining for α1D-AR and kidney injury molecule-1 indicated that α1D-AR was expressed in dedifferentiated proximal tubules of diabetic Zucker rats. Taken together, our results show that α1D-AR expression is upregulated in the diabetic kidneys. PPAR activation suppressed renal expression of α1D-AR in diabetic nephropathy.Alpha In addition to mediating catecholamine-induced vasoconstriction, the α1-ARs in vascular wall have been shown to promote proliferation and hypertrophy of arterial smooth muscle cells and adventitial fibroblasts [α1-AR expression in human prostate, bladder muscle, and smooth muscles has led to the treatment of bladder outlet obstruction and ureteral stones, via blocking these receptors [Alpha species . α1-ARsroblasts , 6. In teceptors , 8.α1-AR subtypes , the α1D-AR has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [α1D-AR knockout models suggest that this α1-AR subtype plays an important role in the overall regulation of blood pressure [α1A- and α1D-ARs are the major functional subtypes of renal α1-ARs in both normal and streptozotocin-induced diabetic Sprague-Dawley rats [Of the three lization –11. Howepressure . Additioley rats .α1-AR subtypes in human kidney cortex [α1-AR mRNA staining was apparent especially in the smooth muscle of arterial walls, whereas weak staining of each of the α1-AR mRNAs was observed in the glomeruli and renal tubules [α1D-AR was most dense in both followed by α1A and α1B [α1D-AR in normal and diabetic rat kidneys remain unknown.Using in situ hybridization, Kurooka et al. identified the gene expression of all three y cortex . They fo tubules . More reand α1B . Howeverα1-AR subtypes in the kidney cortex of normal Zucker lean (ZL) and Zucker diabetic (ZD) rats by microarray and real-time PCR analyses. Confocal immunofluorescence microscopy was performed to evaluate the distribution of α1D-AR in the kidney of Zucker rats. Furthermore, we investigated whether activation of peroxisome proliferator-activated receptor-α (PPARα) or PPARγ, known renoprotective intervention in animal models of type 2 diabetes, would affect renal expression of α1-AR subtypes.In the present study, we examined mRNA levels of three Six-week-old male Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were purchased from Charles River Laboratories . Rats were housed in a temperature-controlled room with a 12 : 12-hour light-dark cycle and free access to Purina 5008 rat chow and water. Blood glucose was monitored using the Accu-chek glucometer by tail-vein blood sampling. In a previous study in which we characterized the time course of blood glucose in this model , we showFor fenofibrate treatment, 12-week-old Zucker rats were divided into 3 experimental groups: vehicle- (0.5% carboxymethylcellulose ig) treated ZL, vehicle-treated ZD, or fenofibrate- (150 mg/kg/day ig) treated ZD (F-ZD) rats for 10 weeks. For rosiglitazone treatment, 12-week-old ZD animals were treated with rosiglitazone (10 mg/kg/day in drinking water) or vehicles for 8 weeks.μm) and rinsed with HBSS. Isolated glomeruli, collected on the 65 μm sieve, were resuspended in HBSS. They were devoid of Bowman's capsule. Tubular fragments were less than 3% of the total number of isolated glomeruli [Glomeruli were isolated from the kidney cortex of ZL and ZD rats at the age of 7, 12, and 20–22 weeks, respectively, by a modified procedure as described previously . Brieflylomeruli .Total RNA was prepared from isolated glomeruli or kidney cortex by using ultrapure TRIzol reagent according to the manufacturer's instructions . The quality of the RNA samples was assessed using the Agilent 2100 Bioanalyzer (G2938A). RNA concentrations were determined spectrophotometrically (absorbance at 260 and 280 nm).α-adrenergic receptor genes among ZL, ZD, and F-ZD groups were identified by a threshold of ≥2 fold change and P ≤ 0.05.Seven hundred and fifty nanograms of total RNA per sample were used for cRNA synthesis and amplification. Agilent Spike-In control RNA was included as an internal control. Cyanine-3-(Cy3-) labeled cRNA was purified and hybridized to Agilent Whole Rat Genome 44k Oligo Microarray chips  according to the manufacturer's instructions. The processed microarrays were scanned with the Agilent G2565BA DNA Microarray Scanner (p/n G2505-A). The scanned images were analyzed with Agilent Feature software (version 9.5.1.1) using default parameters. The resulting text files were loaded into the Agilent GeneSpring GX software (version 7.3) for further analysis. Gene expression values of all datasets were normalized using median normalization. Significantly differentially expressed α1A-, α1B-, and α1D-AR and β-actin gene-specific Taqman probe and primer sets were obtained from Applied Biosystems as Assays-on-Demand (AOD) gene expression products. The AOD identification numbers were Rn00567876 for α1A-AR, Rn01471343 for α1B-AR, Rn00577931 for α1D-AR, and 4331182 for rat β-actin endogenous control. Each sample was run in triplicate, and the comparative threshold cycle (Ct) method was used to quantify fold increase (2t−ΔΔC) compared with controls.Reverse transcription was performed on equal amounts of total RNA by using random hexanucleotide primers to produce a cDNA library for each sample. Real-time PCR reactions were run on an iCycler iQ Real-Time PCR Detection System by using Taqman Universal PCR Master Mix . μm thick cryostat sections of OCT-embedded kidney samples were used. To study the localization of α1D-AR in the rat kidney, the sections were incubated with a mixture of two antibodies overnight: rabbit anti-α1D-AR antibody , mouse anti-α-smooth muscle actin , or goat anti-kidney injury molecule-1 . As a negative control, the sections were exposed to nonimmune IgG (in replacement of primary antibodies) with the same secondary antibodies, and no specific staining occurred. The sections were observed and imaged by Leica confocal microscope.For immunofluorescent staining, 5 t-test was used for comparison between the two groups. Comparisons among multiple groups were performed by one-way ANOVA and Newman-Keuls post hoc test. P < 0.05 was considered statistically significant.Data are expressed as mean ± SEM. Student's α-adrenergic receptor subtypes in the kidney cortex of 22-week-old normal ZL (blood glucose: 108 ± 8 mg/dL) and diabetic ZD (blood glucose: 425 ± 35 mg/dL) rats. The expression profile of each experimental group was determined in three animals per group. α1- and α2-adrenergic receptor subtypes detected in rat kidney tissue. The rank order of expression levels of the three α1-AR mRNAs in normal rat kidney cortex was α1B > α1D > α1A. Among these genes, α1D-AR was significantly upregulated in the kidney cortex of diabetic animals. Compared to normal ZL controls, renal cortical α1D-AR gene was increased by 553% in 22-week-old ZD rats  assay. The relative expression levels of α1A-, α1B-, and α1D-AR mRNAs normalized against β-actin in rat kidney cortex are shown in α1D mRNA was increased by 16-fold in 22-week-old ZD rats compared to ZL controls.To verify the relative transcript levels of α1-AR mRNAs in the diabetic kidneys. Gene microarray analysis revealed that PPARα activation inhibits the upregulation of α1D gene in the diabetic kidneys. Chronic administration of fenofibrate, a PPARα agonist, resulted in a decrease in α1D-AR mRNA by 64% compared to vehicle-treated ZD animals  and cortical (1.7-fold) α1D-AR mRNA levels were significantly higher in the ZD animals, which were further increased by 3.4-fold and 12.9-fold, respectively, at the age of 20–22 weeks.In a previous study , we showα1D-AR gene expression results with its protein level and distribution in the kidney of Zucker animals. As expected, α1D-AR protein was clearly detected in the renal arteries and arterioles in both normal and diabetic animals  was detected in rat kidney cortex, only α1D-AR gene was massively upregulated in the diabetic animals. Moreover, diabetes-related increase in α1D-AR mRNA was inhibited when the ZD rats were treated with fenofibrate or rosiglitazone. Immunostaining for α1D-AR confirmed that intense α1D-AR staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys. Weak α1D-AR protein staining was detected in the glomeruli of normal ZL controls, but there was no obvious staining in the normal tubular epithelium. In consistency with the gene expression results, α1D-AR protein was significantly increased in the glomeruli and proximal tubules of diabetic animals.In this study, the expression and distribution of α1-AR subtype mRNAs has previously been studied in various animal and human organs, and the predominant subtype mRNA expressed differs among species and organs. For example, Kurooka et al. reported that α1A-AR gene was detected more than α1B-AR or α1D-AR in human kidney cortex [α1-AR subtype protein expression in human renal pelvis and calyx tissues and found that α1D-AR was most dense in both followed by α1A- and α1B-AR subtypes, respectively, where the rate of α1B-AR was significantly lower than the other two. In the rat kidney, it was reported that the α1B-AR is predominant when detected by a radioligand binding assay [α1-AR subtype mRNAs (α1B > α1D > α1A) in rat kidney cortex by microarray and qPCR analyses.The expression of y cortex . In conty cortex  recentlyng assay  and RNasng assay . In consα1-AR subtypes, the α1D-AR has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [α1D-AR knockout models suggest that this α1-AR subtype plays an important role in the overall regulation of blood pressure [α1A- and α1D-ARs are the major functional subtypes of renal α1-ARs in both normal and streptozotocin-induced Sprague-Dawley rats [α1-AR subtypes detected in rat kidney cortex, we found that α1D-AR mRNA was markedly increased by 16-fold in the diabetic kidneys. Additionally, diabetes-associated upregulation of α1D-AR mRNA expression was inhibited when the diabetic animals were treated with PPAR agonists, known renoprotective interventions. Therefore, previous evidences and present results strongly suggest that α1D-AR may play an important role in renal physiology and/or pathophysiology.Of the three lization –11. Howepressure . Moreoveley rats . Interesα1D-AR mRNA expression during disease progression in the diabetic animals. We speculate that increased α1D-AR may play a role in the development of diabetic renal hypertrophy and fibrosis. In fact, a role for α1D-AR in vascular hypertrophy and remodeling has been suggested by a recent report that a decrease in the lumen area and an increase in the wall thickness of arteries in hypoxic pulmonary hypertension were strongly inhibited in α1D-AR knockout mice [α1D-AR protein in the kidneys of ZL and ZD rats by immunofluorescence staining. As expected, intense α1D-AR staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys. Compared to low expression level of α1D-AR protein in the glomeruli of normal ZL controls, ZD rats demonstrate a significant increase in α1D-AR signal. Moreover, ZD kidneys displayed strong tubulointerstitial α1D-AR signal within fibrotic areas. α1D-AR protein was expressed in both tubular epithelial cells and interstitial cells. A colocalization of α1D-AR with α-SMA indicates that α1D-AR expressing interstitial cells are myofibroblasts. Future work in the field could be necessary to establish the exact role of α1D-AR in activation and proliferation of renal interstitial fibroblasts.Diabetic nephropathy is characterized by a series of ultrastructural changes, including glomerular and tubular hypertrophy, mesangial expansion, glomerulosclerosis, and tubulointerstitial fibrosis. We have previously demonstrated a progressive loss of renal function in the diabetic animals as evidenced by an increase in urinary protein to creatinine ratio in the ZD rats between the ages of 7 and 20 weeks . In the out mice . In thisα1D-AR expression in tubular epithelial cells of diabetic kidneys. Although there was no obvious staining in the normal tubules, α1D-AR staining was apparent in the dilated tubules in the fibrotic areas. To further characterize α1D-AR expression in renal tubules of diabetic animals, the spatial relationship between tubular α1D-AR and Kim-1, a tubular injury marker, was studied by double staining. In tubulointerstitial fibrotic areas, virtually all dilated tubules expressing α1D-AR were also Kim-1-positive. The selective increased expression by dedifferentiated epithelial cells and activated interstitial fibroblasts supports the potential importance of α1D-AR in renal tubulointerstitial injury. Moreover, recent studies on the pulmonary circulation suggest that catecholamines may participate in the excessive muscularization and fibrosis of the arteries through ERK1/2 signaling pathway in hypoxic pulmonary hypertension [α1D-AR induction in diabetic kidney injury.Another interesting novelfinding in our set of data reported here is the rtension , 20–22. α1-AR subtypes in rat kidney cortex. An increase in renal expression of α1D-AR mRNA and protein was associated with glomerular and tubulointerstitial injury in diabetic nephropathy. Chronic treatment with PPAR agonists prevents the increase in α1D-AR mRNA in the diabetic kidneys. These findings may provide new insights into the prevention and early management of diabetic kidney disease.In summary, the current study highlights mRNA expression of the three


The orientation tuning properties of the non-classical receptive field (nCRF or “surround”) relative to that of the classical receptive field (CRF or “center”) were tested for 119 neurons in the cat primary visual cortex (V1). The stimuli were concentric sinusoidal gratings generated on a computer screen with the center grating presented at an optimal orientation to stimulate the CRF and the surround grating with variable orientations stimulating the nCRF. Based on the presence or absence of surround suppression, measured by the suppression index at the optimal orientation of the cells, we subdivided the neurons into two categories: surround-suppressive (SS) cells and surround-non-suppressive (SN) cells. When stimulated with an optimally oriented grating centered at CRF, the SS cells showed increasing surround suppression when the stimulus grating was expanded beyond the boundary of the CRF, whereas for the SN cells, expanding the stimulus grating beyond the CRF caused no suppression of the center response. For the SS cells, strength of surround suppression was dependent on the relative orientation between CRF and nCRF: an iso-orientation grating over center and surround at the optimal orientation evoked strongest suppression and a surround grating orthogonal to the optimal center grating evoked the weakest or no suppression. By contrast, the SN cells showed slightly increased responses to an iso-orientation stimulus and weak suppression to orthogonal surround gratings. This iso-/orthogonal orientation selectivity between center and surround was analyzed in 22 SN and 97 SS cells, and for the two types of cells, the different center-surround orientation selectivity was dependent on the suppressive strength of the cells. We conclude that SN cells are suitable to detect orientation continuity or similarity between CRF and nCRF, whereas the SS cells are adapted to the detection of discontinuity or differences in orientation between CRF and nCRF. The responses of V1 neurons to visual stimuli presented in the classical receptive field (CRF or center) can be facilitated or suppressed by stimuli in the non-classical-receptive field (nCRF or surround) Previous studies In the experiments presented here, we recorded single-unit activities from the V1 of anesthetized cats and determined the CRF size and suppression index (SI) of the neurons using a size-tuning test. In the experiment, a high-contrast grating, which was drifting at the optimal orientation/direction of the neuron, was varied in size, and the SI of the neuron was measured from the size-tuning curve. We classified the recorded neurons into two categories according to their SI, the “surround-non-suppressive”  bred in the facilities of the Institute of Neuroscience, Chinese Academy of Sciences (Permit Number: SYXK 2009-0066). This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the National Institute of Health. The protocol was specifically approved by the Committee on the Ethics of Animal Experiments of the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (Permit Number: ER-SIBS-621001C). All surgeries were performed under general anesthesia combined with the local application of Lidocaine , and all efforts were made to minimize suffering.2, and rectal temperature were monitored continuously. Anesthesia was considered sufficient when the EEG indicated a permanent sleep-like state. Reflexes, including cornea, eyelid, and withdrawal reflexes, were tested at appropriate intervals. Additional urethane was given immediately if necessary. The nictitating membranes were retracted, and the pupils were dilated. Artificial pupils of 3 mm diameter were used. Contact lenses and additional corrective lenses were applied to focus the retina on a screen during stimulus presentation. At the end of the experiment, the animal was sacrificed by an overdose of barbiturate administered i.v.Acute experiments were performed on 10 cats. Detailed descriptions of the procedures for animal surgery, anesthesia, and recording techniques can be observed in a previous study The recordings were performed with a multi-electrode tungsten array consisting of 22).The monitor was placed 57 cm from the cat's eyes. All cells recorded were obtained from the area of the cortex that represented the central 10° of the visual field. When the single-cell action potentials were isolated, the preferred orientation, drifting direction, spatial frequency and temporal frequency of each cell were determined. Each cell was stimulated monocularly through the dominant eye with the non-dominant eye occluded.The visual stimuli were generated by a Cambridge Systems VSG graphics board. The stimuli were patches of drifting sinusoidal gratings presented on a high-resolution monitor screen  were centered over the receptive field center and randomly presented with different diameters (from 0.1 to 20°). The optimized values for these parameters  were used in these tests. Each grating size was presented for 5–10 cycles of the grating drift, and standard errors were calculated for 3–10 repeats. We defined the CRF size as the aperture size of the peak response diameter  To measure the orientation tuning of the cell's surround, the optimal orientation, aperture, and spatiotemporal frequencies for the center stimulus remained constant. Directly abutting the outer circumference of the center stimulus was a surround grating (with an outer diameter of 20°) of the identical phase and spatial and temporal frequencies. Whereas the center stimulus was maintained at the preferred orientation/direction throughout the experiment, the surround stimulus was shown with variable orientations/directions (in 15° increments). Both the center and surround stimuli were shown at a high contrast (with a range of 20–70% and a mode of 40%). The responses to each patch were recorded for 5–10 cycles of the grating drift, and standard errors were calculated for 3–10 repeats. The cells were classified as “simple” if the first harmonic (F1) of their response to the sine-wave gratings was greater than the mean firing rate (F0) of the response  and the strength of surround suppression were determined using size-tuning tests see . Two patWe initially determined the contrast-response function of the classical receptive field (CRF) for each individual neuron, and then a high-contrast that elicited a response of 90% of the saturation response (with a range of 20–70% and a mode of 40%) was selected to determine the neuron's preferred orientation by stimulating the CRF alone. We then varied the orientation of the surround grating and held the CRF stimuli in the cell's preferred orientation/direction. Altogether, we presented 24 surround orientations/directions shifted in 15° increments.x-axis), and a suppression drop occurred when the surround orientation was 45° away from the preferred central grating. The cell illustrated in θ0 represents the response magnitude (spikes per second) at iso-orientation, Rθ−90 and Rθ+90 represent the response magnitude in the two orthogonal orientations, and Rref is the response to the center stimulus presented alone.We consistently observed that the surround was more selective toward ortho-orientations compared to iso-orientations in the strong suppression neurons than in the weak suppression neurons. To quantify this observation, we used an index for iso-/orthogonal orientation selectivity (OSI). OSI is a measure of the average responses from 2 orthogonal surround orientations relative to the responses to the iso-oriented surround stimuli.OSI directly compared the suppression strength resulting from iso- and ortho-oriented surround stimuli. A value of 1 indicated that the surround had no suppressive effect in ortho-orientations and exerted complete suppression at iso-orientation, a value of 0 indicated that the suppression was equal in magnitude for both iso- and ortho-oriented surround stimuli, and negative values . The remaining cells (46/119) showed a broadly tuned surround or were nearly uniformly suppressive at all orientations.Of the 119 neurons analyzed, there were 22 SN cells and 97 SS cells. The distribution of OSI within the two groups is shown in To assess the overall effects of surround stimulation on the center response, we pooled the data individually for the two categories of cells by normalizing the magnitude of the surround effect to center stimulation alone. Previous studies Levitt and Lund SI) of the neurons. The stronger the surround suppression was, the greater the sensitivity was to the orthogonal orientation between the center and surround, whereas the weaker the surround suppression was, the greater the sensitivity was to the iso-orientation between the center and surround. When both the center and surround were stimulated by the optimal orientation of the cells, the response of the SS cells were maximally suppressed, whereas the response of the SN cells were activated or facilitated. From these results, it can be concluded that SS cells are possibly suitable for the detection of differences or discontinuity in orientations , and conversely, SN cells are most likely adapted to detecting similarities or continuity of orientations .In the present experiments, we recorded 119 neurons from the V1 of cats using high-contrast stimuli. According to the presence or absence of a suppressive surround in the size-tuning tests, we divided these neurons into two categories: surround-suppressive (SS) and surround-non-suppressive (SN). Of the sample we recorded, 82% (97/119) were SS cells with a significant suppressive surround, and 18% (22/119) were SN cells with no suppression or slight facilitation over the surround. We analyzed the relative orientation selectivity between the center and surround using the iso/orthogonal orientation selectivity index (OSI), and observed that the OSI of the surround depended significantly on the suppressive strength  the surround-non-suppressive (SN) neurons tended to detect center-surround continuing orientation and surround-suppressive (SS) neurons possibly to select center-surround discontinuing orientation, and 2) the magnitude of the iso-/orthogonal orientation sensitivity of the surround significantly depended on the suppressive SI of the neurons.


The bell-shaped vibratory angular rate gyro (abbreviated as BVG) is a novel shell vibratory gyroscope, which is inspired by the Chinese traditional bell. It sensitizes angular velocity through the standing wave precession effect. The bell-shaped resonator is a core component of the BVG and looks like the millimeter-grade Chinese traditional bell, such as QianLong Bell and Yongle Bell. It is made of Ni43CrTi, which is a constant modulus alloy. The exciting element, control element and detection element are uniformly distributed and attached to the resonator, respectively. This work presents the design, analysis and experimentation on the BVG. It is most important to analyze the vibratory character of the bell-shaped resonator. The strain equation, internal force and the resonator's equilibrium differential equation are derived in the orthogonal curvilinear coordinate system. When the input angular velocity is existent on the sensitive axis, an analysis of the vibratory character is performed using the theory of thin shells. On this basis, the mode shape function and the simplified second order normal vibration mode dynamical equation are obtained. The coriolis coupling relationship about the primary mode and secondary mode is established. The methods of the signal processing and control loop are presented. Analyzing the impact resistance property of the bell-shaped resonator, which is compared with other shell resonators using the Finite Element Method, demonstrates that BVG has the advantage of a better impact resistance property. A reasonable means of installation and a prototypal gyro are designed. The gyroscopic effect of the BVG is characterized through experiments. Experimental results show that the BVG has not only the advantages of low cost, low power, long work life, high sensitivity, and so on, but, also, of a simple structure and a better impact resistance property for low and medium angular velocity measurements. It senses angular velocity through the standing wave precession effect. The Bell-shaped resonator is a core component of the BVG and looks like the millimeter-level Chinese traditional bell, such as QianLong Bell and Yongle Bell. BVG has not only the advantages of low cost, low power, long work life, high sensitivity, and so on, but also a simple structure and good anti-impact performance for low and medium velocity angular measurements –4.etc. [etc. [The classic vibratory shell solid wave gyro includes: a hemispherical resonator gyro, a cylinder gyro, a ring vibratory gyro, a disc plant vibratory gyro, and so on ,6. Thoseetc. ,8. HRG hc. [etc. –11. The c. [etc. . The cylc. [etc. ,14. Therc. [etc. ; Kristiac. [etc. ; Wu Xuezc. [etc. ,18. A ric. [etc. –22. The c. [etc. . In concetc. Therefore, the project group decided to use the bell structure to make the vibratory gyro, which is named the bell-shaped vibratory angular rate gyro; the resonator is bell-shaped. It is made by a constant modulus alloy, using piezoelectricity to excite and detect. This improves the impact resistance property and maintains the advantages of low cost and high performance. It is desired that the bell structure improves the performance of the gyro. For the novel structure of the gyro, the project group published an article, which describes the design procedure and preliminary experimentation and does not deeply analyze the theory of the BVG. Besides, the angle of the standing wave precession is detected through a capacitance sensor [The proposed bell-shaped vibratory angular rate gyro is inspired by the traditional Chinese bell. Changing the resonator structure of the traditional vibratory gyro improves accuracy and impact resistance. There are many characteristics of the traditional Chinese bell, which includes beautiful sound, vibration stability, a steady mode shape, good impact resistance, e sensor . In the e sensor –27. SincThere are many problems using a bell structure to make a gyro. It is difficult to analyze the bell-shaped resonator, because the vibratory character is very complex and the middle surface is not described using the single linear function. To summarize, the revolution vibratory shell finds a method to solve the vibratory problem of a bell-shaped resonator. For the HRG, VA Matveev and other researchers used the theory of a thin shell to analyze the character of a hemispherical resonator. He presents the equilibrium differential equation and derives the governing equation . This meIn this article, we explore bell-shaped resonator structure and present the design, analysis and experimentation of the BVG. The important work is as follows: to design the structure of a bell-shaped resonator and present the principle of the work, shown in Section 2. In Section 3, a theoretical analysis of the proposed resonator is performed using the theory of thin shells. The strain equation, internal force and resonator's equilibrium differential equation are derived in the orthogonal curvilinear coordinate system. On this basis, the mode shape function and the simplified second order normal vibration mode dynamical equation are obtained. In Section 4, the coriolis coupling relationship about the primary mode and secondary mode is established. The method of the signal processing of the BVG and the control loop of the prototype are presented. In Section 5, to analyze the impact resistance property of the bell-shaped resonator, which is compared with other shell resonators using the Finite Element Method, demonstrates that the BVG has the advantage of better impact resistance. In Section 6, a reasonable method of installation and a prototypal gyro are designed. The gyroscopic effect of the BVG is characterized through experimentation. Experiment results show that the BVG has not only the advantages of low cost, low power, long work life, high sensitivity, and so on, but also a simple structure and better impact resistance for low and medium angular velocity measurements. Finally, some important conclusions are drawn.2.2.1.etc. The technology of multiple surface fusions is very important. However, it has not been explained clearly until now [The core component of the BVG is a bell-shaped resonator, which influences the performance of the gyro directly. The key of the BVG is the structure of the bell-shaped resonator. The bell-shaped resonator is inspired by the traditional Chinese bell, as shown in ntil now . It is cThe bell-shaped resonator looks like the traditional bell, which is designed by a parabolic structure intuitively. In order to reduce the cost and design procedure, the structure of the top, middle and bottom merge into a parabola, and this is achieved to integrate the design. This merge does not change the vibratory character of the structure. The position of the excitation electrode and the detection electrode is very important for the BVG. For the actual bell, to excite bell vibration, one uses a stick to hit the wall of the bell. The vibration effect is very significant. However, the bell-shaped resonator selects the position on the wall of bell. In order to ensure the power consumption and cost of the gyro, it controls the mode shape and detects the precession of the standing wave based on piezoelectricity. The piezoelectric elements are attached to the wall of the bell-shaped resonator to excite the primary mode and detect the second mode. Using the isolated hole reduces the disturbance between neighboring electrodes, as shown in 2.2.ϑ and proportional to the frame rotation angular velocity.The BVG sensitizes angular velocity through the standing wave precession effect. The eight piezoelectric elements are attached to the wall of the bell-shaped resonator uniformly, as shown in During practical application, the stress wave propagates on the resonator, causing the standing wave, and the piezoelectric elements sensitize the stress wave and solve the angular velocity. In order to reduce the influence of piezoelectric elements when the resonator is rotated and to improve the excitation efficiency, the elements should be set near the constrained boundary. Such an excitation type is like the principle of leverage, using a small amount of force to achieve a large deformation to improve the excitation efficiency.The piezoelectric elements chose the PZT5A, which was polarized in the thickness direction. The first and fifth elements contract and expand while applying an alternating current signal in . When th3.For the shell vibratory gyro, the analysis of the resonator vibratory character is very important and focuses on the mode shape function, precession factor and natural frequency. The BVG is a novel structure in the vibratory gyro field and has a unique vibratory character. The analysis of internal force distribution and the equilibrium differential equation are presented using the theory of thin shells. It derives the simplified second normal vibration mode dynamic equation based on the parameters of the gyro.3.1.φ, θ, v), shown in oxyz, the axis of symmetry is axis z. The cross-section is shown in b is the principal radius of the curvature of origin. Define the boundary value of parabolic axis  as a and c, respectively. Then, b can be expressed as b = a2/(2c), and b is twice the focal distance of parabola, b = 2f.The bell-shaped resonator is a parabolical structure and described in the orthogonal curvilinear coordinate system , where φ is the angle between the normal vector of the tangent and the axis of symmetry on the bell-shaped resonator; θ is the circumference angle; v is the thickness of the resonator. The variable basic relationship is as follows in In In the orthogonal curvilinear coordinate system, it is not only convenient to describe the point motion, but also to express the thickness of the resonator. The vibratory character of the bell-shaped resonator is presented in this coordinate.3.2.Based on the theory of thin shells and the Kirchhoff-Lyav hypothesis, the bell-shaped resonator's character as presented includes the relationship of internal force, the equilibrium differential equation and the dynamic equation ,34,35.3.2.1.φ, θ, v). It can be expressed as:u, v, w is the normal distance of three axes; φε, θε is linear strain of the direction, φ and θ, respectively; φθε is shearing strain between φ and θ; φχ, θχ is the change of principal curvature, φk and θk, of the point; φθχ is the change of torsion, φ and θ. The variables are all on the middle surface. When we calculate them, the linear strain on the bell-shaped resonator is expressed as:For the isotropic three-dimensional elastic shell element, the middle surface geometric equation of the bell-shaped resonator is obtained in the orthogonal curvilinear coordinate system,  = h.Based on the transformation relation in the orthogonal curvilinear coordinate system and the expression of internal force in the theory of thin shells, the internal force of the bell-shaped resonator is presented. Using the relations between the mechanical force and stress, one can derive the mechanical membrane forces and bending moments for the bell-shaped resonator –34:(7)F3.2.3.According the the D'Alembert principle, if the sum of force and inertial force and the torque are all equal to zero, which is applied to the isolated unit, then the unit is in a state of equilibrium. The bell-shaped resonator is fit for this principle. As far as we know, the middle surface of the bell-shaped resonator cannot stretch. The three components of tangential displacement in the shell bending equation are equal to zero. It can be expressed as :(13)ɛφ=The normal force and shearing force are equal to zero:φq, θq, qv is the middle surface load of the bell-shaped resonator, which is in relation to the input angular velocity. For the point, P , on the bell-shaped resonator, the motion expression in the vector is expressed as:φ̂, θ̂, v̂ is the unit vector of the coordinate axis. The motion vector, R, of the point, P, is as follows:Therefore, we can derive the equilibrium differential equation , V (φ), W (φ) is the Rayleigh functions about the bell-shaped resonator's second order intrinsic vibration mode on the three axes, respectively. p (t), q (t) is the displacement of the vibratory rigid axis.To solve the According to nA is the coefficient of the vibratory amplitude; n is the order of the resonator vibration. According to the p (t) and q (t). Calculating using Maple finds that the equation is too complex to solve using the traditional method. For the HRG, the equation is easier than the BVG, which is solved through the Bubnov-Galerkin method [Substituting n method -38. The m0, b, c0, β is an undetermined function about φ, θ, h and other material parameters. Therefore, the natural frequency of the bell-shaped resonator is:For The precession factor is:The typical analytical method does not solve this complex problem, but it clearly describes the vibratory procedure, the force and equilibrium relation. In practice, the Finite Element Method is widely used for the bell-shaped resonator to analyze the natural frequency, mode shape and work mode.3.4.L1 = 21 mm, R1 = 11 mm, H1 = 0.7 mm, L2 = 9 mm, L3 = 15 mm; as shown in Analysis of the vibratory character is based on the theory of thin shells. It is very necessary to know the overall mode shape of the bell-shaped resonator. The modal simulation is presented using FEM. The main structure parameters of the resonator are as follows: Using the mode shape function of Section 3.3, the simulation result is shown in 4.The mode shape control about the standing wave on the circumference is very important for the BVG. p and q. p is the axis of the primary mode, and q is the axis of the second mode [p and q. In np is the control axis of p (first and fifth element); pp is the sensitive axis of p (third and seventh element); nq is the control axis of q (second and sixth element); pq is the sensitive axis of q (fourth and eighth element). The relationship of p and q is described in K is the precession factor; n is the mode order of the resonator; pf is the force applied to p; qf is the force applied to q; Ω is the angular velocity; pω is the frequency of the primary mode; qω is the frequency of the secondary mode; ωθ is the angle between the primary axis and p; τθ is the angle between the damping axis and p; pτ, qτ is the delay time of the axis; The transversal surface of the standing wave is the vibratory ring. On the ring, there are two rigid axes, ond mode ,42. CoriThe diagram of the BVG signal process method is shown in 5.The bell-shaped vibratory angular rate gyro's greatest strength is its remarkable impact resistance property. Using FEM to compare the hemispherical resonator gyro , the cylThe maximum stress of HRG is 181 MPa; the cylinder vibratory gyro is 1,600 MPa; the novel ring vibratory gyro is 3,000 MPa; the BVG is 151 MPa. The result shows that the bell-shaped resonator has the best impact resistance property. Based on the mechanics of materials, the tensile strength of Ni43CrTi is 500 MPa. Therefore, the bell-shaped resonator does not get damaged in 20,000 × g. During the impact process, the resonator produces a small deformation. It is quickly restored after the impact. The control loop also can restrain the influence of the small deformation, too.6.6.1.Step1: The piezoelectric element with the wire attached on the wall of the bell-shaped resonator;Step2: The fixed axis fastens the bell-shaped resonator to the foundation;Step3: The wire connects to the insulated joint on the foundation.The main structure of the BVG includes the bell-shaped resonator, a fixed axis and a foundation. These connect together through mechanical technology, as shown in Finally, the 9 wires connected to the signal process system include 8 wires of piezoelectric elements and GND. The wires connect to the circuit board through the insulated joint, which is installed on the foundation. The prototypal bell-shaped resonator is shown in 6.2.The experiment of the bell-shaped resonator includes: natural frequency test, mode shape test, coriolis test and gyroscopic effect test. The natural frequency and mode shape have been tested in . In this6.2.1.ppV = 10V) is applied to the first and fifth element. In order to readout the signal clearly, the output signal of the piezoelectric element is processed by the operator amplitude and summed by the piezoelectric element with the same axis. In the test, the Lissajous pattern is a good method to analyze the coriolis effect about the two axes, p and q. The oscilloscope that was chosen is the Tektronix TDS 3032B. Channel 1 connects to the pp axis, used as x. Channel 2 connects the pq axis, used as y. When Ω = 0, the picture is as shown in The sine alternating current signal (During the test, the bell-shaped resonator is in an uncontrolled situation. The frequency split of the tested bell-shaped resonator is 0.5 Hz. In the static state, the two axes exit the vibratory coupling. This described Lissajous pattern is not the line. When the angular velocity is applied to the sensitive axis, the couple effect is very obvious and the standing wave produces precession. The bell-shaped resonator is already sensitive to the coriolis effect.6.2.2.The prototypal BVG includes power, a signal sample board and a signal processing board, as shown in The manufacturing technology has imperfections. Specifically, the piezoelectric element is difficult to stick on the resonator with a curved surface of variable curvature.There is no temperature compensation for the bell-shaped resonator. The long work time leads to a change of the frequency and influences the drift. The bell-shaped resonators vibratory characteristics change as the temperature changes.Structure processing error and control loop error exist. Improving the method of restraining the frequency split and designing the control loop is needed.The parameters of the governing equation of the bell-shaped resonator are not exact.The BVG is fixed on the high-precision angular velocity turntable to test the gyroscopic effect. The results are shown in etc. The performance of a prototypal BVG is not satisfactory, but the method of design and analysis is demonstrated perfect.In the future, important work is needed: improving the manufacturing technology and the control loop design, designing a temperature compensation method, 7.Based on the orthogonal curvilinear coordinate system, the equilibrium differential equation using the theory of thin shells is presented, and the motion of the point on the bell-shaped resonator is described. The vibratory character of the resonator is analyzed with the input angular velocity. On this basis, the mode shape function and the simplified second order normal vibration mode dynamical equation are obtained. The Coriolis coupling relationship about the primary mode and the secondary mode is established. The method of signal processing of the BVG and the control loop of the prototype is presented. To analyze the impact resistance property of the bell-shaped resonator, which is compared with the other shell resonator using the Finite Element Method, demonstrates that the BVG has the advantage of better impact resistance property. A reasonable means of installation and the prototypal gyro are designed. The gyroscopic effect of the BVG is characterized through experimentation. The experimental results shows that the zero drift instability is 22.5°/h and linearity is 1.24%. The index is poorer than other shell vibratory gyros. The main reason is: the manufacturing technology has imperfections; there is not any temperature compensation; structure processing error and control loop error exist; the parameters of the governing equation of the bell-shaped resonator are not exact. In the future, important work includes improvement of the performance of the BVG. In conclusion, the BVG has not only the advantages of low cost, low power, long work life, high sensitivity, and so on, but also a simple structure and better impact resistance for low and medium angular velocity measurements.


There is limited research data on post-rehabilitation function and quality of life despite the increasing role of rehabilitation in the care of stroke patients in Malaysia. Outcome data is important in evaluating the effectiveness of stroke rehabilitation services in the country.The aim of this study was to assess function and quality of life in stroke patients following intensive rehabilitation at a tertiary hospital.This was a cross-sectional study of 91 stroke patients; mean age 58.9±10.6 years, 79% male, median stroke duration 13 months who have completed intensive individual rehabilitation at the Universiti Kebangsaan Malaysia Medical Centre in the years 2010 and 2011. Rehabilitation outcome was measured with the use of standardised tools; Rivermead Mobility Scale (RMI), Berg’s Balance Scale (BBS), Sit to Stand Test (STS) for lower limb strength and Timed 10 metre walk test for walking speed. Post-rehabilitation disability level and quality of life were also assessed on a Modified Rankin Scale (mRS) and Euro-Qol 5 Dimensions-Visual analogue Scale (EQ5D-VAS), respectively. All data were analysed descriptively using SPSS version 18.The median duration of rehabilitation was 10.5 months (range 5-25) in the study patients and post-rehabilitation mean mRS was 2.3±0.7. The median RMI was 13 (range 6-15), median BBS 51 (range 20-56) and median STS 15.5 secs (range 7.9-83.9 secs). The EQ5D-VAS mean score was 71.5±17 and mean walking speed at the completion of intensive rehabilitation was 49.4±28.3 m/min; less 22 m/min when compared with the optimum walking speed required for safe road crossing.Although our stroke patients gained satisfactory levels of mobility, balance and strength following intensive rehabilitation, they have not achieved optimum speed of walking to enable effective community ambulation. Prolongation of rehabilitation programme may assist in further functional and quality of life gain among the post-stroke patients.


Introduction. Chronic obstructive pulmonary disease (COPD) is associated with comorbidities such as cardiovascular disease, metabolic disease, osteoporosis, and anxiety and/or depression. Although pulmonary rehabilitation programs are proven to be beneficial in patients with COPD, it is unclear whether comorbidities influence pulmonary rehabilitation outcomes. The aim of the present review was to investigate to what extent the presence of comorbidities can affect pulmonary rehabilitation outcomes. Methods. The systematic literature search  resulted in 4 articles meeting the inclusion criteria. The odds ratios  of the logistic regression analyses, with comorbidities as independent variables and pulmonary rehabilitation outcomes  as dependent variables, were used for data extraction. Results. Patients with anxiety and/or depression less likely improve in dyspnea. Osteoporosis is associated with less improvements in functional exercise capacity, while cardiovascular disease does not seem to negatively impact on this outcome. Patients with cardiovascular comorbidity will experience less positive changes in quality of life. Conclusion. Evidence from literature suggests that comorbidities can have a negative influence on pulmonary rehabilitation outcomes. Screening for comorbidities in pulmonary rehabilitation settings seems useful to readdress the right patients for individually tailored pulmonary rehabilitation. Chronic obstructive pulmonary disease (COPD) is often described as a multicomponent syndrome, characterized by pulmonary and extrapulmonary consequences –3. The pThis systematic review adhered where possible to the PRISMA  statement on developing a systematic review .Studies meeting the following criteria were included. (1) The study included stable patients with COPD (GOLDI-IV). (2) The patients with COPD should be screened in terms of comorbidities before starting pulmonary rehabilitation or comorbidities should be collected from medical records. (3) Comorbidities should include cardiovascular disease  and/or metabolic disease  and/or osteoporosis, and/or anxiety and/or depression. (4) The study predicted the outcomes of standard outpatient rehabilitation programs, that is, dyspnea, exercise capacity, or quality of life. Reviews were not selected, but references were hand-searched for relevant literature.We did not report muscle weakness as a comorbidity in the present review as it can be assumed that muscle weakness is most likely present in those patients referred for pulmonary rehabilitation.A systematic electronic literature search was performed in Pubmed, PEDro (Physiotherapy Evidence Database), and EMBASE (Excerpta Medica dataBASE). The search terms we used included COPD, comorbidities or extrapulmonary comorbidities, and rehabilitation. In PEDro, the search was performed by indicating COPD, fitness training, and cardiothoracics in the proposed key terms. A more detailed description of the search strategies is depicted in β-coefficient (standard error (SE)) from the logistic regression analysis, we calculated the odds ratio (OR) with the formula: OR = eβ. The lower limit of the 95% CI of the odds ratio was then found based on β − 1.96∗SE and the upper limit of the 95% CI based on β + 1.96∗SE. When only the β-coefficient without SE was presented, the author was contacted to become the 95% CI of the OR. A significant OR < 1 indicates that the presence of the specific comorbidity leads to a lower chance of improving in the outcome investigated. With a significant OR being higher than 1, it is more likely that the presence of the comorbidity leads to improvement in the outcome of pulmonary rehabilitation.From each article, we extracted the study design, the type of analysis and the disease severity. The content of the rehabilitation program described in each article was used. The specific comorbidities, their prevalence and the outcomes of pulmonary rehabilitation were extracted and the odds ratio ) from each logistic regression analysis was retrieved to investigate the impact of comorbidities on outcomes of pulmonary rehabilitation. If the odds ratio was not calculated, but the author did report the n = 2962) came from one study [symptoms of dyspnea [functional exercise capacity were 4 times lower in patients with osteoporosis [quality of life [The systematic review resulted in 4 articles, involving a total of 3595 stable patients with COPD. The majority of patients , measured by the Borg scale before performing a 6MWD test. Both anxiety and depression were associated with more symptoms of dyspnea at baseline, measured by the Baseline Dyspnea Index  and after the 6MWD . Vanfleteren and colleagues [β = −0.11; P = 0.007). Mentz et al. [r = −0.34; P = 0.008). The present review could not draw clear conclusions about the influence of metabolic disease on functional exercise capacity. However, Sava et al. [The present study contains several limitations. First of all, the majority of studies were retrospective, which could have impacted on the accuracy of collecting comorbidities. Another limitation was that we only included studies reporting odds ratios to show associations and studies providing data on improvements in pulmonary rehabilitation outcomes. Therefore, studies looking at comorbidities and outcomes in a different way were not withheld , 21–25. lleagues  rejectedz et al.  focused a et al.  focused a et al. . In anota et al.  found pa2, continuous training) to moderate  to high  to very high intensity  to improve exercise capacity. The chosen intensity depends on the preserved ejection fraction and the exercise capacity of the patient during the pretraining period, measured by a cardiopulmonary exercise test [2max) [2max), during sessions of 20–30 minutes for a period of 8–14 weeks [Focusing on comorbidities in patients with COPD is of importance since they contribute to the overall severity of the disease and have a negative impact on patient's life expectancy , 26. Conise test . Patientise test . The guit [2max) , 36. Litt [2max) . For pat14 weeks . Accordi14 weeks  the inte14 weeks . These lComorbidities are prevalent in patients with COPD and they potentially have a negative effect on outcomes of standard pulmonary rehabilitation. More specifically, they could reduce the benefits in terms of dyspnea, functional exercise capacity, and quality of life. Based on the present review, we conclude that including patients with comorbidities in pulmonary rehabilitation programs is still reasonable as they improve with training. However, we should be aware that, without altering the program, response rates will be lower. An optimal treatment should therefore include a baseline assessment of comorbidities with a subsequent individually tailored pulmonary rehabilitation program.


Purpose. To describe healthcare consumption and costs prior to, during, and after multidisciplinary rehabilitation due to arthritis. Methods. 306 patients (age 18–75 years) with arthritis scheduled for multidisciplinary rehabilitation care in 9 rehabilitation centres and 4 rheumatology hospital departments were included and followed for 6 months. Costs were estimated in Euros (€) for the total sample and five clinical subgroups. Results. Healthcare costs ranged from €3,033 to €91,336 and were significantly higher at hospital departments compared to rehabilitation centres: €9,722  and €4,250 . While total costs prior to and after rehabilitation were stable for those receiving rehabilitation at a hospital, there was a significant increase in costs for those being at a rehabilitation centre. Total mean costs were more than doubled when including social costs: from €32,410  to €51,491 . Conclusions. Healthcare and social costs for arthritis rehabilitation were substantial both before and after a rehabilitation stay. It is important to explore methods to reduce the length of rehabilitation stay and production loss connected to rehabilitation of patients with rheumatic disease. Despite advances in pharmacological treatment, work disability among individuals with chronic arthritis is substantial , and nonIn Norway, multidisciplinary rehabilitation care for patients with inflammatory or noninflammatory arthritis is available in rheumatology departments at national, and regional hospitals and in specialised rehabilitation centres. Rheumatology hospital departments offer multidisciplinary rehabilitation care through in- and outpatient programmes, and the teams usually consist of several types of health professionals, for example, rheumatologist, nurse, physical therapist, occupational therapist, social worker, and psychologist. The teams in the rehabilitation centres are often limited to rheumatologist, nurse, and physical therapist, and hence, there is a less costly health service. There exists no unanimous practice for referring arthritis patients in need of rehabilitation care to a hospital or a rehabilitation centre. However, since patients referred to a rehabilitation centre must be able to take care of basic self care activities, it is expected that these patients have a lower level of disease severity, for example, as measured by physical function outcomes. Similarly, it is expected that patients, who can attend outpatient rehabilitation programmes at hospitals, have less activity limitations than patients in need of inpatient multidisciplinary rehabilitation care. €) 2047 versus €1111 in inpatient/outpatient, respectively. Other observational studies of inflammatory arthritis patients in hospitals report that total costs are significantly influenced by severity of the disease, such as level of pain, presence of rheumatoid factor, and grip strength [A few previous studies have shown that multidisciplinary rehabilitation care, provided in outpatient rehabilitation programmes, has significantly lower costs than inpatient programmes –5. In anstrength –12, few The purpose of this study was to describe healthcare consumption and related costs over a period 3 months prior to, during, and 6 months after receiving multidisciplinary rehabilitation care for various clinical subgroups of patients with inflammatory or noninflammatory arthritis. A second aim was to investigate the effect of diagnosis, clinical setting, age, gender, severity of disease, comorbidity, and work status on total healthcare costs. We expected that patients with arthritis had a reduced need for healthcare in the first months following multidisciplinary rehabilitation care of at least one week duration. Further, we expected that the healthcare costs were higher by higher age, higher severity of disease , for patients who were on sick leave, and higher among patients referred to a rehabilitation stay in a hospital as compared to a rehabilitation centre. This was a multicenter, longitudinal observational study in which all institutions/hospital units, which provided multidisciplinary rehabilitation care, in South-East Norway were invited to participate. There were 15 rehabilitation centres and 4 rheumatology units in hospital eligible, of which 9 specialised rehabilitation centres and the 4 rheumatology hospital departments agreed to participate in the study. Eligible patients at the participating centres were consecutively recruited over a three-month period from September to December in 2006 and followed in 6 months after discharge.All patients aged 18 to 75 years with an inflammatory joint disease or osteoarthritis of any location and scheduled for a rehabilitation stay of at least one week were invited to participate in the study. Patients diagnosis was confirmed by a rheumatologist at each of the clinical sites. Exclusion criteria were serious psychiatric comorbidity or inability to communicate in written Norwegian. The patients were recruited by research assistants at the institutions/departments, who provided information about the study to the patients, including informed agreement and written consent. The study was approved by the regional committee for medical research ethics.At admission physicians examined the patients and provided diagnostic information. The patients filled in a comprehensive questionnaire at admission and discharge and a postal questionnaire at six-month follow-up. The baseline questionnaire included sociodemographic and health status variables. Sociodemographic variables concerned age, gender, level of education, work status, co-morbidity (number of diseases from a list of 12 possible diagnostic groups), use of medication , and use of assistive tools. Health outcomes were the Modified Health Assessment Questionnaire (MHAQ)  and the Use of health care services during the 3-month period before the rehabilitation stay was collected in the baseline questionnaire before the patients started their rehabilitation. Use of health care services in the 6-month follow-up was recorded by a monthly cost diary, including registrations of number of visits to a general practitioner, physical or manual therapist, medical specialist, social worker, and alternative therapist, number of days of hospitalization and/or rehabilitation, use of medication (both on prescription and over the counter medication), and number of days of sick leave from work. Different types of healthcare costs are presented in four cost groups: cost group 1 considers costs for primary healthcare services, cost group 2 for secondary healthcare services, cost group 3 covers use of medication, and cost group 4 provides costs for production loss for employed patients. The four cost groups are presented in  Information on cost per unit was collected from different sources . Costs p€3,513* . When the DRG group was impossible to define, the unit cost was based on an average DRG per day. The rehabilitation length was calculated as number of days between admission and discharge, including the weekends (7 days per week). Costs incurred at the hospital were based on diagnosis-related groups (DRGs) 2006. DRG is an international coding system, used for administrating both clinical and financial activity, in the specialist health care. In Norway there are about 500 DRG groups. All DRG groups are given a specific weight to reflect the treatment intensity relative to the average patient. The cost for a DRG group is estimated by multiplying the cost weight for that specific DRG group with the cost of one DRG. In 2006 the cost of one DRG was €) with 1 € = 8 NOK.Unit cost for the different types of treatment  was based on charges. For the group “others” we used the lowest charge of the other treatments as an estimate. Medication costs were based on the price list from the Norwegian Medicines Agency. The costs of acupuncture, homeopathy, and other types of treatments were based on out of pocket cost reported by the patients. The cost of sick leaves (for those who were employed) is estimated to be equal to average income inclusive social costs . We thus€15. However, there is an upper limit for out-of-pocket costs of approximately €250 for Norwegian people. Exceptions are alternative treatments and treatment procedures not referred by a medical doctor, which have to be covered in total by the patient him/herself. In Norway, the social security system covers almost all costs related to hospital stay, including the costs for medication and other treatments during the stay, and the costs related to production loss. Furthermore, the social security system covers the main costs for rehabilitation stay and use of primary care health services for people with a rheumatic disease diagnoses, whereas there is a minor out-of-pocket payment for these services. The out-of-pocket payment for a stay at the rehabilitation institutions is in average Continuous variables are presented by means with standard deviations and categorical variables are presented by frequencies and percentages for the total sample and for each of the diagnostic subgroups. As the cost data were highly skewed, we present the total costs with both median (with min-max) and mean with standard deviation. Cost estimates within the subgroups are presented with mean and confidence intervals, which were estimated with the bootstrap method. For differences in trends within the cost groups and between clinical subgroups, we used rank sum based on “Kruskal-Wallis equality-of-populations rank test” according to the clinical subgroups and time periods, respectively. When assessing associations between the total costs and potential predictors, we used the total healthcare costs in groups 1, 2, and 3 for the rehabilitation stay and the 6-months follow-up as main outcome. The effects of clinical subgroup, age, gender, comorbidity, working status, baseline scores of the MHAQ, SF36 physical and mental function, and use of assistive tool were tested separately in the inflammatory and non-inflammatory subgroups by means of Ordinary Least Square regressions. Statistical analysis was performed with SPSS 14.0 and STATA 10.5% level of significance was used.Of 581 eligible patients a total of 460 (72.2%) accepted to participate in the study. There was incomplete diagnostic data for 3 patients, 23 patients withdrew before they started the rehabilitation, and 70 patients dropped out in the period between discharge from the rehabilitation stay and 6 months of follow-up. Of the 367 responders to 6-month follow-up 61 patients had lacking or incomplete data for the use of healthcare, leaving a total of 306 patients for current analyses. There were no significant differences between the initial included study sample and the final set of 306 responders with regard to diagnostic group, clinical setting, gender, work status, co-morbidity, and baseline physical function . The non responders, however, were slightly younger, were more frequently single, and had lower level of education and poorer mental health scores according to the SF36 Mental health.n = 60) or rehabilitation centre (n = 60). A minor subgroup of 14 patients had rehabilitation due to inflammatory arthritis in an outpatient clinic at one of the hospitals. Patients at rehabilitation centres and hospital departments differed significantly with regard to several of the sociodemographic variables at baseline: for example, patients at the rehabilitation centres were older, more likely to be female, and less likely to be employed and had more co-morbid conditions. Patients in the hospitals, however, used more inflammatory medications and also tended to use more assistive tools. There was, however, no statistical significant difference between the five subgroups with regard to use of assistive tools and use of relaxing/sleeping medication. The mean age was 61 years (SD 9.6), 75% were female, and the majority had an old age- or disability-related pension (68%) . The majP < .001)  than at the rehabilitation institutions  ( < .001) . At base < .001) . The patMean costs related to health consumption in the primary healthcare services (cost group 1), secondary healthcare services (cost group 2), use of medication (cost group 3), and production loss (cost group 4) for each of the five subgroups prior to, during, and after the rehabilitation stay are presented in Healthcare consumption and costs during the 3-month period prior to and 6-month period after multidisciplinary rehabilitation care were similar with no significant changes within the two groups with inflammatory arthritis treated at a hospital. For the three clinical subgroups receiving rehabilitation at a rehabilitation centre, there was a significant increase in mean costs due to healthcare consumption in the primary care, whereas there was a significant decrease in mean costs of secondary healthcare services for the two osteoarthritis subgroups. There was also a significant decrease in use of medication in the postsurgery osteoarthritis subgroup across the time periods. Furthermore, the mean costs due to medication use were higher among patients with an inflammatory diagnosis as compared to patients with osteoarthritis within all the time periods. €32,410  to €51,491 , respectively  and the inflammatory patients at inpatient hospital departments incurred more than twice healthcare costs than the other three clinical subgroups  ranged from ubgroups . When su€39,624 . As MHAQ, SF36 Physical function, SF36 Mental health, and assistive tools all are variables indicating health status, we estimated five different models including different variables  from inclusion in the multidisciplinary team rehab stay until 6-month follow-up was mean €13,000 during rehabilitation and 6 months after the rehabilitation stay. Female gender, use of assistive tools, and poor physical function in terms of either MHAQ or SF36 Physical function also had a significant effect on the healthcare costs. The correlation between the MHAQ and use of assistive tools could explain why MHAQ did not have a significant effect in Models 1 and 3. In these models, age, work status, comorbidity, and mental function did not have an effect on use of health care services among patients with inflammatory arthritis.For patients with inflammatory disease all the models showed that the total costs in a hospital unit were significant more costly than rehabilitation in a hospital outpatient and at a rehabilitation centre . Being iFor the patients with osteoarthritis there was no significant difference in total costs across the other three clinical subgroups at the rehabilitation centres . Only fe€3,033 to €91,336 and were significantly higher at hospital departments compared to rehabilitation centres with mean €9,722  and €4,250 , respectively. Second, while total costs prior to and after rehabilitation were stable for those receiving rehabilitation at a hospital, there was a significant increase in costs for the three clinical subgroups at a rehabilitation centre. Third, increased total healthcare costs were associated with having rehabilitation in an inpatient hospital department, being a women, using assistive tools, and having reduced physical function in patients with inflammatory arthritis, whereas only female gender influenced healthcare costs among patients with non-inflammatory arthritis. These findings indicate that—for chronic conditions like rheumatic diseases—it is important to look at healthcare consumption and related costs in a wider time perspective than only the rehabilitation period. This study provides a descriptive analysis of direct and indirect costs incurred by various subgroups of patients with inflammatory or non-inflammatory arthritis receiving multidisciplinary rehabilitation care. Three main findings were observed: first, the total healthcare costs ranged from €6,000 and €2,400 for undifferentiated arthritis. In a more recent review of cost-of-illness studies from 2007 the yearly cost estimates of rheumatoid arthritis ranged from 11,717 to 28,498 Canadian Dollars (CAD) depending on the method [In general, vast cost discrepancies have been reported across studies  and acroe method . Overall, in-patient hospital costs accounted for the largest proportion of direct healthcare costs, whereas the costs related to primary healthcare and medication accounted for lower costs. Even though the rehabilitation stay at hospitals was significantly shorter than in rehabilitation institutions (16 versus 23 days), the costs for rehabilitation were significantly higher at the hospitals. The main reason for this difference is the large difference in the price per day/night per person in hospitals as compared to rehabilitation centres. This finding is in line with our a priori expectations and also with several previous studies , 18 thatWe expected that the healthcare costs prior to a rehabilitation stay were higher as compared to the period after rehabilitation, in particular in the first 3-month period after the rehabilitation stay. An underlying assumption was that patients with arthritis had a reduced need for healthcare in the first months following multidisciplinary rehabilitation care of at least one-week duration. Opposite to our expectations, the healthcare costs were surprisingly high during the whole observation period, in particular for the three clinical subgroups receiving rehabilitation at a rehabilitation centre. Some of the increased costs right after the rehabilitation stay might have been a consequence of interventions that were initiated during the rehabilitation stay followed up in the primary care after discharge. It can also be argued that six months of follow-up was too short time to show that the healthcare costs might have reached the level prior to the rehabilitation stay. On the other hand, similar to the findings in our study a recently published observational study from Germany  reported€2,494 , the costs estimated in our study were about fourfold. Since comprehensive inpatient and outpatient multidisciplinary rehabilitation program have been found to be equally effective in patients with rheumatoid arthritis, outpatient programmes are recommended [The mean direct cost of outpatient rehabilitation in our study was about doubled compared to the German study  and was ommended –5. Both ommended  suggest The costs due to production loss associated with arthritis exceeded by far the direct costs of healthcare. Although this finding is similar to previous reviews of cost-of-illness studies for patients with arthritis , 19, wheAnother limitation is the lower number of patients in the outpatient group, which must be considered when interpreting the results within this group. As mentioned above, one might argue that 6-month follow-up was short and a longer follow-up period could have helped to explore whether costs due to healthcare consumption in the primary care decreased back to the level prior to a rehabilitation stay. Furthermore, the fact that the response rate at 6 months of 66% and nonresponders were slightly younger and more frequently single, with lower level of education and poorer mental health, might indicate a potential for selection bias. High rates of nonrespondents among younger people have also been reported in other studies using mailed surveys , and it The main strengths of this study were a large sample size, further data for all study institutions were concurrently collected, and investigation of the use of healthcare as well as production loss after the rehabilitation stay was performed in a detailed way. We also consider that this study has implications for further research. The observed large differences in costs across clinical settings in which rehabilitation is provided should be tested out in a randomised, controlled trial in order to assess whether the increased costs can be justified with increased effectiveness. Moreover, it is important to investigate whether a good follow-up procedure in the primary care can prevent or reduce the need for costly inpatient multidisciplinary rehabilitation. Finally, the study results can be used in a discussion regarding what is optimal level of healthcare consumption and/or rehabilitation in various subgroups of patients with arthritis. The patients in this study had a lower level of healthcare consumption prior to the rehabilitation period than afterwards. That might seem contradictory as one would assume that their condition was more severely affected before than after. It is crucial to understand the relationship between need and/or use of healthcare consumption and severity of chronic diseases, in order to develop longer-term optimal disease management plans.To conclude, our data demonstrated substantial costs related to multidisciplinary rehabilitation care and use of healthcare consumption among patients with arthritis, before, during, and after the rehabilitation stay. The time period following a scheduled rehabilitation stay was characterized by surprisingly high healthcare consumption and costs in patients with arthritis. Both the clinical setting in which rehabilitation was provided and disease severity itself had a large impact on healthcare consumption and costs. In patients still working, indirect costs in terms of sick leave were considerable. As a clinical implication, methods to reduce the length of rehabilitation stay should be developed, in particular at hospitals, and regarding production loss with a focus on cost saving, given that health outcomes are constant. Clinical studies on different rehabilitation strategies should include health-economic analyses contributing to the best management of rheumatic diseases.