C. elegans UBR-1 leads to a specific motor deficit: when adult animals generate reversal movements, A-class motor neurons exhibit synchronized activation, preventing body bending. This motor deficit is rescued by removing GOT-1, a transaminase that converts aspartate to glutamate. Both UBR-1 and GOT-1 are expressed and critically required in premotor interneurons of the reversal motor circuit to regulate the motor pattern. ubr-1 and got-1 mutants exhibit elevated and decreased glutamate level, respectively. These results raise an intriguing possibility that UBR proteins regulate glutamate metabolism, which is critical for neuronal development and signaling.UBR1 is an E3 ubiquitin ligase best known for its ability to target protein degradation by the N-end rule. The physiological functions of UBR family proteins, however, remain not fully understood. We found that the functional loss of  UBR1 exhibit severe systemic defects, but the biology behinds UBR1’s physiological function remains elusive. Here we found that the C. elegans UBR-1 regulates glutamate level. When UBR-1 is defective, C. elegans exhibits increased glutamate; this leads to synchronization of motor neuron activity, hence defective locomotion when animals reach adulthood. UBR1-mediated glutamate metabolism may contribute to the physiological defects of UBR1 mutations.Ubiquitin-mediated protein degradation is central to diverse biological processes. The selection of substrates for degradation is carried out by the E3 ubiquitin ligases, which target specific groups of proteins for ubiquitination. The human genome encodes hundreds of E3 ligases; many exhibit sequence conservation across animal species, including one such ligase called UBR1. Patients carrying mutations in  In eukaryotic cells, the ubiquitin-proteasome system mediates selective protein degradation , 2. E3 uubr1 mutants exhibit less efficient chromatin separation, and mildly increased doubling time [C. elegans UBR-1 results in delayed degradation of a regulator for post-embryonic hypodermic cell division, but does not cause obvious hypodermic defect [JBS), a genetic disorder with multi-systemic symptoms including pancreatic insufficiency, growth retardation, and cognitive impairments [JBS pathophysiology remains elusive [UBR1 acts not only as an E3 ligase for the N-end rule substrates, whose metabolic stability is determined by the identity and post-translational status of their N-terminal moiety, but also for substrates that do not harbor the N-terminal degrons , 7. The ing time . Yeast Uing time . The losc defect . In mammc defect . The simc defect . In humaairments . To date elusive .C. elegans has a single UBR1 ortholog, UBR-1. Using this simplified animal model, we reveal that the functional loss of UBR-1 leads to a specific, late onset and prominent motor pattern change, and such a change is reversed by removing a metabolic enzyme, GOT-1, which we find to synthesize glutamate from aspartate.Glutamate is an abundant amino acid. As a metabolite, it is essential for metabolism and development. As a neurotransmitter, glutamate-mediated signaling regulates animals’ motor and cognitive functions –18. As subr-1 and got-1 mutants suggests that UBR-1 may affect glutamate metabolism. Consistent with this notion, we found that both genes are critically required in premotor interneurons to affect the reversal motor pattern change. Further, our metabolomics analyses reveal an inversely correlated change—increased and decreased glutamate level—in ubr-1 and got-1 mutant animals, respectively. These findings reveal a previously unknown role for the UBR family protein in glutamate metabolism. They further allude to the possibility that a common cellular defect, such as that in glutamate metabolism, may contribute to UBR’s multi-systemic functions.The genetic interaction between C. elegans generates movements through propagating body bends. In a genetic screen for mutants with altered motor patterns, we isolated hp684 . We compared their body curvature and duration of reversals during these events : a recessive and nonsense mutation that leads to truncation of the last 194 amino acids (Q1864X) of UBR-1 ; hp821 a N-terminal four base pair deletion that leads to a frame-shift and a premature, N-terminal stop codon (E34X), and hp821hp833, in addition to hp821, a seven-base-pair deletion in the RING finger that leads to a frame shift and premature internal stop codon (E1315X). All alleles, like hp684, are recessive and viable, all exhibited motor defects similar to hp684, including reduced bending, increased duration, and reduced initiation frequency during reversal movements , the reversal motor defects  did not  exerted the strongest partial rescue (Prig-3), or RIM (Pgcy-13) alone, led to modest to no rescue  (What underlies the reduced bending in  (A-MNs) . Multipl (A-MNs)  in freelConsistent with the notion that A-MNs execute reversal movements, they exhibited calcium changes when animals moved backwards  at the pyridoxal phosphate-binding domain in GOT-1.2 . GOT enzymes catalyze the transfer of an amino group between aspartate and α-ketoglutarate (α-KG) and convert them to oxaloacetate (OAA) and glutamate [We identified the causative mutation in  GOT-1.2 , one of lutamate , 49 , but not in all muscles (Pmyo-3), fully reverted the motor pattern of ubr-1; got-1 to the reduced bending as in ubr-1 mutants . As previously reported [C. elegans , whereas the aspartate level exhibited a massive increase of 27.3 ± 4.76 folds    analyses on whole worm lysates. Indeed, got-1 mutants exhibited increased AMP/ATP and NADP/NADPH, and decreased GSH/GSSG glutathione ratios . In ubr-1; got-1 mutants, similar to got-1 mutants, the glutamate level was reduced by 21.6 ± 2.4% , whereas the aspartate level was increased by 30.2 ± 5.3 folds    . Interesanimals) , which me either .ubr-1’s motor defects, we surveyed their levels in ubr-1, got-1 and ubr-1; got-1 adults by LC-MS/MS. Unlike the case for glutamate, which exhibited inversely correlated changes between ubr-1 and got-1 mutants, and between ubr-1 and ubr-1; got-1 mutants, the α-KG and OAA levels did not exhibit consistent, or correlated changes  [Because most of the critically required premotor interneurons for UBR-1’s role in motor pattern are cholinergic , the devr-1-RFP)  that allr-1-RFP)  that labr-1-RFP)  Fig 7)..Pnmr-1-Rubr-1 mutants, both reporters exhibited prominent and moderate decrease in fluorescence intensity in premotor interneurons AVA and AVE, respectively . Such a decrease was not observed for other interneurons including RIM , was similar between wildtype and ubr-1 mutants  mutants was the glutamate level. Cumulatively, these results strongly support that the primary metabolic change—the level of glutamate—causes the motor pattern change in ubr-1 and ubr-1; got-1 mutants.Our metabolomics analyses of AA, α-KG , and allAA, α-KG . Becauseubr-1 and ubr-1; got-1 mutants. We reckon that other UBR-1-expressing neurons likely also contribute to such an alteration. Furthermore, the glutamate change in other neurons and cells may cause other physiological effects that were not examined in this study, where we only followed the most obvious motor defect.Our analyses measured glutamate level in whole animals, not the specific neuron groups that are critically required for both UBR-1 and GOT-1’s effect on reversals. Examples of genes with broad expression, but cell-type specific functions have been reported , 54, 55.Glutamate has multiple functions. Being a key neurotransmitter, glutamate also serves as the sole precursor of another neurotransmitter GABA. Glutamate is also one of the abundant amino acids partaking in other metabolic pathways including the amino acid synthesis, energy production and urea cycle. An elevation of glutamate not only affects signaling in glutamatergic and likely GABAergic neurons, but also increases metabolic stress in all cells.ubr-1 mutants. Interestingly, within the reversal motor circuit, premotor interneurons that exhibited the prominent changes, AVA and AVE, provide direct synaptic inputs to the A-class motor neurons. These observations suggest that compared to other cells and neurons, the development and function of premotor interneurons, in particular AVA and AVE, may be more susceptible to glutamate increase, likely through both increased glutamatergic inputs and cellular stress.Because both UBR-1 and GOT-1 exert strong effects on the reversal motor pattern through premotor interneurons, most of which cholinergic , the devubr-1’s motor defects through neurons. These results indicate that maintaining glutamate level in neurons, especially the premotor interneurons, is critical for the proper motor output of ubr-1 mutants.Despite its broad expression, GOT-1 can only influence In the mammalian brains, glutamate has to be locally synthesized because it cannot cross the blood-brain barrier . The actC. elegans neurons have remained elusive [The main sources of glutamate synthesis and homeostasis in  elusive . Our stuHow UBR-1 negatively regulates glutamate level remains to be elucidated. UBR family proteins are often addressed in the context of N-end rule E3 ligases , 7, 10. C. elegans, the CED-3 caspase promotes apoptosis. During postembryonic development, it interacts with UBR-1 to expose the N-degron of LIN-28 [ubr-1 mutants’ bending defects , integrated multi-copy arrays (hpIs), single-copy integrated arrays (hpSi), and a got-1.2 GFP knock-in allele (hp). Transgenic animals carrying extra-chromosomal arrays (hpEx) were created by co-injecting the DNA plasmid of interest and a co-injection marker at 5–30 ng/μL. Extra-chromosomal arrays were integrated into the genome using UV irradiation to create stable, transgenic lines (hpIs) [got-1 tissue-specific rescue lines were generated using Mos1-mediated single copy insertion (hpSi) as previously described [Transgenic strains include those with extra-chromosomal multi-copy arrays (s (hpIs) . We founescribed . All inthp881) was generated by CRISPR-Cas9 mediated homologous recombination, following protocols described in [got-1 start codon, the entire got-1 coding sequence, and 1.5kb sequence downstream of the got-1 stop codon. GFP was inserted in-frame after the last amino acid of GOT-1. A LoxP-Prps-27-NeoR-loxP cassette was inserted between the stop codon and 3’ UTR. After injection, animals were allowed to lay eggs overnight at 25°C before G418 selection. Animals with extra-chromosomal arrays were selected against as described. Candidates for insertion were confirmed by genotyping and sequencing. Confirmed insertion lines were injected with a Pelt-3::Cre plasmid to remove the LoxP cassette. The resulting hp881 allele was outcrossed against N2 wildtype animals twice before crossed into the ubr-1 mutants.The in-frame GOT-1 C-terminal GFP fusion allele , an aversive stimulus for C. elegans [2 solution was pipetted into a small circle (~20 mm in diameter) in the middle of the plate for the animal to roam. One animal was placed in the center of the circle and allowed to habituate for one minute prior to recording for three minutes. For the next recording, CuCl2 was pipetted onto the same circle before another animal was placed on the plate and recorded. For all data presented in the same graph, animals were recorded on the same day, with at least one animal from each genotype recorded on the same plate. We altered the order of recording for animals of different genotype.When transferred to a new, thinly seeded plate, escribed , 35 mm N elegans . BrieflyBehavior was recorded using a Zeiss Axioskop 2 Plus equipped with an ASI MS-40000 motorized stage and a CCD camera (Hamamatsu Orca-R2). Tracking and analysis were performed using Micromanager and Image J software plug-ins developed in-house (courtesy of Dr. Taizo Kawano). Image sequences were sampled at 100-msec exposure (10 frames per second). For the post-imaging analysis, an Image J plug-in was used to skeletonize the worm and extract its centerline. The centerline was divided into 32 equal segments, and the angle between each segment and its tangent line was calculated to quantify the curvature of the animal. These segments were binned into four groups to capture the most consistent curvature trends across the entire length of the animal. The directionality of movement (forward vs. backward) was determined by first identifying the anterior-posterior axis or the “head” and “tail” points, which was manually defined at the first two frames and verified throughout the recording. To calculate directionality of movement, the displacement of the midline point in relation to the head and tail for each worm was determined based on its position in the field-of-view and the stage coordinates. Image sequences wherein animals touched the edge of the recording field and crossed over on themselves were not processed.Analyses of the output data were carried out using an R program-based code developed in-house (courtesy of Dr. Michelle Po). From a group of recordings (N≥10 for each genotype per experiment), we quantified the following parameters, among other behavioral parameters analyzed by the program: 1) Curvature, the average curvature of each animal at each segment; 2) Initiation  Duration . Body curvature, initiations, and durations were calculated for forward and backward locomotion separately. Curvature value during reversals were analyzed from backward movements over 5 seconds.hpIs460 (Punc-4-GCaMP6::wCherry), using a calcium reporter GCaMP6 fused in-frame at its C-terminus with wCherry for both tracking and ratiometric correction for motion artifacts in moving animals during recordings [C. elegans were placed on a 2.5% agar pad, immersed in a few drops of M9 buffer, and covered by a coverslip to allow slow crawling. Each recording lasted for 3 minutes. Images were captured using a 40x objective on a Zeiss Axioskop 2 Plus equipped with an ASI MS-40000 motorized stage, a dual-view beam splitter (Photometrics) and a CCD camera (Hamamatsu Orca-R2). The fluorescence excitation light source from X-CITE (EXFO Photonic Solution Inc.) was reduced to prevent saturation of imaging field. The fluorescent images were split by Dual-View with a GFP/RFP filter set onto the CCD camera operated by Micromanager. The 4x-binned images were obtained at 50-msec exposure time (10 frames per second). Regions of interest (ROIs), containing the soma of DA7, VA10 and VA11, were defined and simultaneously tracked using an in-house developed Image J plug-in [Animals of various genotypes were crossed into a reporter cordings . Briefly plug-in . The ratThe pair-wise phase relationships between the activity patterns of A-MNs during each reversal event were assessed by cross-correlation analyses. In each example trace, cross-correlation during the entire period of reversal was calculated to determine the phase lags between the calcium signals of VA11, DA7, and VA10. The maximum of the cross-correlation function denotes the time point when the two signals are best aligned; the corresponding argument of the maximum correlation values denotes the lag between the two neurons. The absolute time lags were used to represent the synchrony of VA11, DA7 and VA10’s activation. N (≥10) refers to the number of reversal events analyzed for each genotype per experiment.°C at 1400 rpm. These samples were centrifuged at 14000 rpm for 10 min at 4°C. Supernatant was transferred to fresh tubes and lyophilized in a CentreVap concentrator (Labconco) at 4°C. Samples were stored at −80 °C until used for HPLC or LC-MS/MS analyses.Synchronized last-stage larval worms were grown on 100 mm NGM agar plates seeded with OP50 bacteria. Worms were collected using M9 buffer and were washed thoroughly to remove bacteria. Worm pellets were snap-frozen in liquid nitrogen and pulverized using a cell crusher. Amino acids and other metabolites were extracted by addition of ice-cold extraction solvent  and incubated on dry ice for an hour with occasional thawing and vortexing. Samples were then moved to a thermo mixer (Eppendorf) and shook for an hour at 4Amino acid quantitation was performed using the Waters Pico-Tag System (Waters). After hydrolysis and pre-column derivatization of the sample by PITC, samples were analyzed by reverse phase HPLC . LC-MS/MS metabolite analysis was performed as described previously . Raw valg for 10 minutes to remove insoluble materials. The supernatant was used for the AST assay and pellets were used to quantify the total protein in the samples (as described above).Synchronized late L4 larval stage worms were grown on 100 mm NGM agar plates seeded with OP50 bacteria. Worms were collected using M9 buffer and were washed thoroughly to remove bacterial contamination. Samples were homogenized by sonication in 100-200ul ice-cold AST buffer. Samples were centrifuged at 13,000GOT  activity was measured using a colorimetric assay with AST Activity Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Data were normalized by the total protein content of the whole worm lysate as determined by a Bradford Protein Assay.C. elegans were grown on 100 mm NGM plates seeded with OP50 and collected using M9 buffer. Lysates were prepared as described previously [Mixed stage eviously . For wesFor bending curvature and calcium imaging analyses, statistical significance was determined using the Kruskal-Wallis test and the two-way repeated measures (RM) ANOVA and subsequent post-hoc analysis. For metabolite analyses, two-tailed Student’s t-tests were applied to determine statistical differences. p< 0.05 were considered to be statically significant. All statistics were performed using Prism software (GraphPad).S1 Figubr-1 mutants during reversals (left to right panels). The wildtype animal generates sinusoidal body bends, whereas ubr-1 animals do not bend. This defect was rescued by restoring the expression of UBR-1. Dots denote position of tail. Scale bar: 200μm. In ubr-1(hp820) (B) and ubr-1(hp821) (E) alleles (grey line), bending curvature is reduced throughout head to tail compared to wild type (black line), and this was rescued by restoring expression of UBR-1 using its endogenous promoter (red line). ubr-1 mutants have fewer initiations  and longer durations for reversals . ***P<0.001, **P<0.01, *P< 0.05 by the Two-way RM ANOVA test. Data are represented as mean ± SEM. ***P<0.001, **P<0.01, *P<0.05 by the Kruskal-Wallis test. Data are represented as mean ± SEM.A) Representative images of wildtype animals and two alleles of (TIF)Click here for additional data file.S2 FigPnmr-1-RFP reporter (red). B) A confocal image of animals expressing endogenous GOT-1::GFP from the knock-in allele (green), and the Pnmr-1-RFP reporter (red). Robust expression of UBR-1 and GOT-1 was present at the pharynx; neuronal UBR-1::GFP and GOT-1::GFP signals were present in the AVA, AVE and RIM premotor interneurons (denoted), and other unidentified neurons (not shown). Scale bar, 5 μm.A) A confocal image of animals expressing a functional UBR-1::GFP transgene from its endogenous promoter (green), and the (TIF)Click here for additional data file.S3 Figubr-1 mutants, the reversal duration is increased, while reversal initiation frequency is decreased. Both the parameters were rescued by restoring the expression of UBR-1 in premotor interneurons, but not in GABAergic or cholinergic motor neurons. C-D) Expression of UBR-1 in multiple premotor interneurons, which include AVE/RIM exhibited significant rescue of ubr-1 for reversal duration and initiation frequency. Expression of UBR-1 in the RIM alone or in AVA alone did not result in rescue. *P<0.05, **P<0.01, ***P<0.001 by the Kruskal-Wallis test. Data are represented as mean ± SEM.A-B) In (TIF)Click here for additional data file.S4 Figgot-1 restores the reversal duration (A) and the reversal initiation frequency (B) in ubr-1 mutants. Restoring GOT-1 expression in multiple premotor interneurons, including AVE and RIM, reverted the reversal duration of ubr-1; got-1 to that of ubr-1 (A), whereas the reversal initiation frequency exhibited the trend of decrease but did not reach statistic significance (B). Expression of GOT-1 in RIM alone had no effect, while the expression of GOT-1 in AVA partially reverted the reversal duration. *P<0.05, **P<0.01, ***P<0.001 by the Kruskal-Wallis test. Data are represented as mean ± SEM.The functional loss of (TIF)Click here for additional data file.S5 FigMetabolite levels measured by LC-MS, normalized against the total protein level in the lysate. All mutants were normalized to that of wildtype animals. Metabolites of the TCA cycle did not show any coordinated changes among these mutants.(TIF)Click here for additional data file.S6 Figubr-1 animals, which were labeled with cytosolic RFP, from the L1 juvenile larva to adult stages. Depending on the focal planes, some images contain that of nuclei, which were devoid of RFP signals. Left panels: images from wildtype animals. The surface of both somata was round and smooth throughout larval development and in young adults. Right panels: images from ubr-1 mutants. In young (L1 to L3) larva, somata appeared similar to those in age-matched wildtype animals. In the L4 larva and adult animals, somata developed rough surface and short branches, denoted by arrows. B) The RIM soma exhibited normal morphology in adults, similar to wildtype animals. C) The round morphology AVA and AVE somata in ubr-1 adults were restored in ubr-1; got-1 double mutant adults, and in ubr-1 adults with restored UBR-1 expression in multiple premotor interneurons including AVE and RIM. Asterisks (*) mark the AVA or AVE axons, which could not be shown in some panels when they were in a different focal plane as the somata. Scale bar, 2μm (for the L1 and L2 panels) 5 μm (for the L3 to adult panels).A) Confocal images of the AVA and AVE premotor interneuron somata in wildtype and (TIF)Click here for additional data file.S1 Movieubr-1 mutant exhibits prominent rigidness during reversals.The (MOV)Click here for additional data file.S2 Movieubr-1 mutant background led to significant improvement of body bending during reversals.The functional loss of GOT-1.2 in the (MOV)Click here for additional data file.S3 Movieubr-1 mutants did not improve body bending during reverses.The functional loss of the LIN-28 protein in the (MOV)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The recovery of hand function is one of the most challenging topics in stroke rehabilitation. Although the robot-assisted therapy has got some good results in the latest decades, the development of hand rehabilitation robotics is left behind. Existing reviews of hand rehabilitation robotics focus either on the mechanical design on designers' view or on the training paradigms on the clinicians' view, while these two parts are interconnected and both important for designers and clinicians. In this review, we explore the current literature surrounding hand rehabilitation robots, to help designers make better choices among varied components and thus promoting the application of hand rehabilitation robots. An overview of hand rehabilitation robotics is provided in this paper firstly, to give a general view of the relationship between subjects, rehabilitation theories, hand rehabilitation robots, and its evaluation. Secondly, the state of the art hand rehabilitation robotics is introduced in detail according to the classification of the hardware system and the training paradigm. As a result, the discussion gives available arguments behind the classification and comprehensive overview of hand rehabilitation robotics. Stroke, caused by death of brain cells as a result of blockage of a blood vessel supplying the brain (ischemic stroke) or bleeding into or around the brain (hemorrhagic stroke), is a serious medical emergency . Stroke The human hands are very complex and versatile. Researches show that the relationship between the distal upper limb  function and the ability to perform ADL is stronger than the other limbs –9. The dRobot-assisted therapy for poststroke rehabilitation is a new kind of physical therapy, through which patients practice their paretic limb by resorting to or resisting the force offered by the robots . For exaExisting reviews of hand rehabilitation robotics on poststroke motor recovery are insufficient, for most studies research on the application of robot-assisted therapy on other limbs instead of the hand . FurtherThis paper focuses on the application of robot-assisted therapy on hand rehabilitation, giving an overview of hand rehabilitation robotics from the hardware systems to the training paradigms in current designs, for a comprehensive understanding is pretty meaningful to the development of an effective rehabilitation robotic system. The second section provides a general view of the robots in the entire rehabilitation robotic system. Then, the third section sums up and classifies hardware systems and the training paradigms in several crucial aspects on the author's view. Last, the state of the art hand rehabilitation robotics is discussed and possible direction of future robotics in hand rehabilitation is predicted.Robot-assisted poststroke therapy for the upper extremity dates back to the 1990s . It has Now, the hand rehabilitation robotics has been greatly developed with the rapid development of neurosciences and clinic knowledge, which makes the design of hand rehabilitation robotics for poststroke rehabilitation become more complex for involving multidisciplinary knowledge such as anatomy, neurosciences, cognitive and learning science, and rich experience in the clinic , 26–28. Motor impairment of stroke patients differs from person to person, and states and conditions vary in the course of recovery. Factors such as types of muscle state , phases of stroke , and levels of stroke (from mild to severe stroke) should all be taken into consideration for individualized treatment . For exaAlthough current theories underlying the motor recovery are insufficient, a few of existing rehabilitation theories are still instructive and significant for the application of robot-assisted therapy. Three main popular directions are the theories of neurophysiology, neurodevelopment, and motor learning. The neurophysiology mechanism, such as the plasticity and compensatory function, is the theoretical basis of the poststroke motor recovery . The neuThe application of hand rehabilitation robotics is to achieve effective rehabilitation by making a serial of decisions on the designing of both hardware system and the training paradigm , 23, 26.The evaluation of rehabilitation robotics reflects the recovery effects of human functions, which make it imperative for a complete design. There are varied ways on evaluating the robots in current researches, including the performance on improving range of motion (ROM) of joints, the velocity of motion, the force exerting to hands, and the executing of functional tasks. A set of classic clinical scales is also used to evaluate the design by measuring the recovery of stroke patients. The most frequently used scales include the Arm Research Assessment Test (ARAT), Fugl-Meyer Assessment (FMA), and Motor Activity Log (MAL) . It shouThe requirement of hand rehabilitation robotics is not the same as the traditional design of robots, because it involves humans. Some special matters like medical principles and applications in clinic need to be paid attention to, for example, the safety and the availability of hand rehabilitation robots. Although some other requirements such as participation are shown to be meaningful, these may not be essential for every kind of robot-assisted therapy. Two of the most imperative requirements are introduced here.With the participation of human, safety becomes the top priority for the design of rehabilitation robotic hands. Any mechanical problems would do serious harm to the human bodies because of the close contact between the robotic hand and the human hands . TherefoThe motor recovery training is long term and costly to most patients. Some researches show that the main limitation to the practical use of hand rehabilitation robotics is the availability . The avaThere exist many kinds of ways for the classification of hand rehabilitation robots, some of which follow the convention in mechanical design (focus on the hardware system), while others follow the convention in rehabilitation (which focus on the training paradigms) , 26. In The hardware system is the foundation of hand rehabilitation robots. It decides the possible types of motion the robots can offer to the patients and the possible signals the robots can obtain from patients. The hardware system can be classified in details in a mechanical classification . HoweverExisting hand rehabilitation robots can be divided into two main types according to the alignment of the device and the user: the end effector and the exoskeleton. The end effector is external to the patients' body while the exoskeleton is worn by human beings .The end effector is external to the body of patients, and it provides required force to the end of the user's extremity to help or resist the motion . For exaDifferent from the end effector, exoskeleton device can be worn on the body of patients. The joint and links of the robot have direct correspondence with the human joints and limbs, respectively . For exaThe function of actuation is to transform different kinds of energy to actuate the motion of robots. There are 5 kinds of actuation mentioned here: elector motor, hydraulic, pneumatic, pneumatic muscle, and human muscle. Although there are still some other kinds of actuation such as the piezoelectric and the shape memory alloy which are promising for being thin and lightweight, they are not mentioned here for either being limited by their own technical dilemma in practical application or just being theoretical design –64.The electrical motor is almost the most widely used actuation in design of hand rehabilitation robots, because they are easily available, reliable, and easy to control and with high precision. For example, the HANDEXOS designed by Chiri A et al. is actuated by the force transmitted for DC motor to a Bowden cables; the exoskeleton hand robotic training device designed by Ho et al. is actuated by micro linear electrical motor , 58. TheThe pneumatic actuators are used much less than the electrical motor in hand rehabilitation robots, such as the ASSIST designed by Sasaki et al. This actuator has advantages such as less requirement of maintenance and can be stopped under a load without causing damages , 65. AltThe development of pneumatic artificial muscle makes the actuation another choice. The pneumatic muscle made of rubber inner tube with a shell can inflate or contract. For example, the commercial hand rehabilitation robotic system produced by Kinetic Muscles Inc. (USA) is actuated by air muscle actuator . There iThe hydraulic actuators are very good in performance such as can generate higher torque compared with the electric or pneumatic systems and can be controlled in high precision and frequency . But, thThe contralateral extremity can be thought as an actuation too. The hand rehabilitation robots actuated by the bilateral limb are usually used in the robotic system applying the bilateral training strategies . The robThe human muscle on the impaired hand can be activated by functional electrical stimulation (FES) to complete the motion of impaired hand the same as robotic actuation . MoreoveOther designs that corresponded with the active training modalities may not provide actuations, but totally actuated by patients' own hand; this means for a high requirement to patients' residual motor abilities . AnotherThe function of transmission is to transform the motion of actuator into a desired direction to complete the execution of a hand's motion. Most of these are a consequence of the choices of actuator or the mechanism.Linkages are popular choices in the hand rehabilitation robot system, the same as in the traditional mechanical design. The linkages are light, convenient, and can be easily controlled in a given trajectory. On the one hand, the problem of coincidence of the rotational axis can be settled by using the cross-joint structure. On the other hand, the complexity of device can be reduced according to the concept of fDOF by using the linkage structure . For exaThe cable is also frequently used as the transmission in the hand rehabilitation robots, including the pulley cable and Bowden cable. The pulley requires a continuous tension to maintain traction on the pulleys, which limits the use , 79. On Although the hand rehabilitation robot system has some effects just according to the continuous passive motion (CMP) concept, the participation of patients seems to be more effective in the rehabilitation system , 83. ThiThe sensors detecting physical signal such as the force and position (or motion) are the most used sensors in the robot system of hand rehabilitation . The funThe other kinds of sensors detecting the bioelectrical signals such as the EEG or EMG signals are also frequently used in the hand rehabilitation robot system , 41. TheThe training paradigms designed according to the rehabilitation theories or clinical results with good efficacy might be the most crucial things in the motor recovery. The essence of training paradigms is how the robots interact with the patients from signal perception to physical contact. To date, no comprehensive reviews discussing the training paradigms are found, although some classifications such as training modalities and training strategies have been widely used in researches , 23. ForThe classification of training modalities relates to conventional therapy modes used in a clinical practice  14]. It. It14]. In resistive training modality, the patient completes the motion under the resistive force offered by the robot. This kind of training mainly focuses on the force training, so the robot usually adopts the impedance control schemes , 92. ForIn active training modality, the patient executes the motion on his own ability, while the function of robots is being used as a measurement device to offer feedback to the patients. In this kind of training modality, the motion is totally controlled by human, while no control scheme is needed here. For example, Adamovich et al. studied the rehabilitation system in which the patients wore CyberGloves and CyberGrasp device to get the position and force information. When the patients execute motion on their own, the real-time information of the hand are collected and presented with a virtual scene and feedback force , 98. In In assistive training modality, the patient executes voluntary motion of his hand with the assist of the robots. The robot offers continuous force to the hand during the motion. This kind of training modality can be as simple as the HandSOME designed by Brokaw et al., in which the assistant force is offered by a spring to counteract the muscular tension . No contIn active-assistive  training modality, the patient completes the motion with the help of robots, but here, the robot would not offer force to the hand until the patient cannot move on its own. The active-assistive training modality is the most widely used one in the rehabilitation robot system , 104. AcIn passive training modality, the motion of the patient's hand totally depends on the force offered by the robots. According to the continuous passive motion (CPM) concept, the massive passive training would promote the motor recovery. Robots of passive training modality are usually controlled by a position to do repetitive training, in which the robots move the hand from the start position to the end position and then move back , 108. ThAccording to the collected papers, current design of movements in hand rehabilitation robots can be divided into two patterns. The first pattern focuses on the range of motion, muscle strength, and spasticity while the second one focuses on performance of functional tasks, especially for the ADLs. This is corresponding to the International Classification of Functioning, Disability and Health (ICF) framework , in whicMost of current design adopts the movement on functional level, that is, movements that focus on increasing the joint range of motion, improving muscle strength, and decreasing spasticity and so on. Intensively executing simple motion on one or several joints by such as continuously grasp or extension of palm is usually used in this kind of training , 112. MoMovement on activity level can usually be found in task-specific training or training of activities of daily living (ADL) , 114–116The differentiation between different training paradigms is essentially decided by different human-robot interactions which are also called training strategies in other papers. Human-robot interaction is essential for the motor recovery, because in rehabilitation robotic system, the subject of human is the key point, which is often inadequately considered. This is different from traditional industrial or field robots that execute motion automatically or with the explicit command from humans, because the humans are both the commander and a component of the robot system of hand rehabilitation . For humA number of innovations combined methods such as action observation or motor imagery with rehabilitation robotics. These different combinations could be generalized according to the different patterns of inductions. The induction is used to induce motor intention from the patient. It seems that the better induction pattern should be able to induce stronger motor intention and inspire more motivation of the motion from patients , 40. TheThe task-specific (or task-oriented) induction usually offers real-life objects with which the patients perform motion of daily activities such as grabbing a bottle  or pickiThe mirror induction refers to the mirror therapy or bilateral training . In traditional mirror therapy, by using a mirror that is positioned orthogonally in front of the patient, the reflection of the right arm in the mirror provides an illusion that the left arm is being moved . The mirThe virtual induction refers to virtual objects presented by computers . There aThe efficiency of game paradigm is promoted by findings that show immersive training, or implicit practice can strengthen the learning ability of patients , 129. ItIn a normal pathway of healthy subjects, the motor intention can be conducted to the muscle in electrical signal to actuate motion of the hand directly, while in a pathway of stroke subjects, the motor intention has to be detected by the robots and then the robots actuate the motion of the hand. The intention can be detected from the brain, muscle, and both the paretic hand and the contralateral healthy hand.The EEG signals are represented brain signals, which contain the motor intention that can be detected. The EEG signal is the most convenient signal in the brain-computer interface (BCI) system. The BCI technology has been greatly developed in decades and has many effective applications in the robot system of hand rehabilitation. The application of BCI technology is mainly based on the fact that the motor imaginary (MI) promotes the recovery of limbs. The effectiveness of MI in rehabilitation has been demonstrated long before. Researches show that through the MI of limb movements, the feeling of paretic limb would be aroused to some extent for the motor cortex would be activated during MI, though the patients did no relevant motion –137. ComBiebaumer is the earliest one to demonstrate that stroke patients can evoke MI-related ERD/ERS as the health individuals , 140. UnThe electromyography signal of stroke patients is not enough to drive the motion of paretic hand, but it is strong enough for the data collection instruments to collect, thus it provides a new method of control for rehabilitation robotic hands, which is to control the motion of robotic hand with processed signals collected from the muscle. Researchers like Ho et al. designed electromyography- (EMG-) driven hand robots –145. AftThe EMG technology detects motor intentions directly from the EMG signal of patients, to help the execution of initiative motion. That is why it has better effects than the traditional passive training without the participation of muscles. However, there are still some points that should be focused for EMG-driven rehabilitation robotics. First, the EMG signal varies in the different conditions of stroke patients, so the rehabilitation robotic system should be adaptive . Second,One of the most direct methods of sensing the user's intention is to measure the force exerted by the patient at the interface . This meAlthough the human has its own proprioception, it is either abnormal or too weak for stroke patients. The robot can offer extra feedback to the brain to form an integrated close-loop pathway, which is meaningful for the human-robot interaction. The feedback is usually an augmented compensation for an attenuation of haptic feedback , 153. ItThe purpose of using the feedback of real-time state is to compensate the absent self-feedbacks such as haptic feedback of the hand. The feedback can be the visualization of force exerted to the device, index of EMG/EMG signals, or joint motion compensator realized by actual mechanism , 154–156The feedback of motor performance is mainly used in a game paradigm. The efficiency of this feedback might be explained by the simple Pavlovian conditioning. The award of completing a motion presented by a computer program would reinforce the motor feedback loop and inspire more motivations in the next motion . NeverthAs the classification shows, varied kinds of design of hand rehabilitation robots have been explored in all these studies, thus making the hand rehabilitation robotics being greatly developed in recent years. However, the progression of clinical use of hand rehabilitation robots has not caught up with the pace of the designing of the prototype. The result of an evidenced-based analysis even shows that many other alternative interventions have “stronger evidence” than the robot-assisted therapy . Here, tOne obvious general limitation of current researches is that many researches ignore the importance of evaluating the design of hand rehabilitation robots, which could be found from A solution is that more standard randomized controlled trials (RCTs) should be designed on evaluating current robot system of hand rehabilitation. On the one hand, the approach of using clinical scale which is easy to administer for assessment should be popularized in hand rehabilitation robotic design, for it is the most acceptable way in clinical use now. On the other hand, to set other standardized evaluation methods realized by more objectives with the help of robots might be promising, for it overcome the biggest disadvantage of clinical scale of being subjective for the visual scores of the test , 171. ThLacking of pertinence is another limitation of the current design. It can be found from The function of a specific HRR can often be realized in several different combinations of different hardwares. What decides the efficacy of HRR is not a single hardware but the whole hardware system that makes choosing of hardware not just finding an absolutely best hardware but trading off different components under a specific need of application.The exoskeleton robots are the dominant choice compared with end effector robots under the trend of in-home treatment. Although the end effector has the advantage in omitting the problem of the coincidence of joints, this merit can also be realized by underactuated exoskeleton robots based on the notion of fDOF. Besides, a lot of expense can be reduced, because the controlled DOF can be decreased according to the fDOF notion. The promotion of in-home treatment makes the application of end effector robots fewer in hand rehabilitation because of constraints on portability.Choosing of actuation is mainly between the electrical motor and the pneumatic artificial muscle. The merits that like being easy to control, with high precision and with high energy-to-weight, make the electrical motor the prior actuation in hand rehabilitation robot. Being portable and small in volume are very crucial for designing a flexible robotic hand. The development of soft-bodied robots makes the pneumatic actuation another noticeable choice in robotic hand. The robots using pneumatic artificial muscle can be small and with high safety compared with traditional actuations, while the application of soft-bodied robots for hand rehabilitation is still limited by the extra gasholder.Among the choice of actuations, the contralateral extremity and the human muscle should be more focused on. The mirror therapy, although with undefined mechanism, has already gotten good performance under many practical uses in conventional therapy and several initiatory applications in robot-assisted therapy , 166. ItApart from soft body structure, the linkages are almost the necessary among the transmissions. Using the linkages to form an underactuated robot is the mainstream method in solving the problem of joint coincidence. Although the soft body structure has the advantage in being adaptive to human bodies, the complexity on control system and the problem of portability limit its application. It should be mentioned that the jointless robots designed by In et al. using the cable provide new thoughts in using cable to solve the motion problem .The extensive use of sensors for bioelectrical signal is the tendency in design of hand rehabilitation robot. Although the sensor is not necessary for a robotic hand, the consensus on the importance of patient's participation demands sensors for detecting signal from human bodies. The bioelectrical signals that were obtained directly from the delicate neural activities expend the methods of detecting signal form the patients. Even though the patients cannot move their hands, the bioelectrical signal can also be used in place of the physical signal. Endeavor on exploring the sensors for bioelectrical signal should also focus on solving the problem of lacking of effective features for control signals, because the motion of the hand is varied.Choosing of training paradigms decides the kernel of a hand rehabilitation robot, while it has not been fully introduced in papers combined with the whole rehabilitation robot system in consideration of patient difference and the merit and demerit of different designs.Choosing of training modalities should better be nonconstant in the whole period of training for maximizing the efficiency of hand rehabilitation robots, in correspondence with the practical condition of patients. Two demerits for current choosing of training modalities: First, in passive motion, the absence of patients' active participation slows down the process of motor relearning; while, second, in positive motion, most patients in early stage are too weak on both muscle strength and mental activity, thus making the training process hardly inaccessible for them. Adopting varied modalities as the state changes in the rehabilitation may make a difference. For example, use passive training in the early stage of treatment for patients with difficulty in generating detectable intention to restore the muscular tone; use active-assistive training in the midstage of treatment to promote the process of motor relearning; and use active training in the later stage of treatment for patients to regain independent motor ability in case of overreliance. Besides, the control scheme is better adopted according to different training modalities. For example, for the resistive training modality, the impedance control is a good choice, because the impedance control can allow more dynamic interaction with the patients. Fuzzy controlled may be a good choice for active-assistive training, because the participation of intention detected from biosignal can be better classified in this control scheme , 173. ItMovement on activity level would be the focus in subsequent studies. It could be found in Human-robot interaction should be taken more into consideration for it decides the basic mode of the robot's role in motor recovery of patients' brain, and it connects the hardware system with the training paradigms. This is provided in First, the system in most designs is not real-time but has been provided through long time-window sequence. This makes the hand rehabilitation robot system a not real closing loop system and separates the perception of execution and feedback of patients , 176. FoSecond, the humans have varied intentions while current researches could only figure little of them. The more closing the intention is detected from the central nervous system, the littler intention could be detected, while more intention can be detected from the distant extremity. Besides, current technologies on detection intention from the brain or muscle are either low in accuracy or inconvenient in clinical use. On the one hand, better detection system should be developed. On the other hand, although motions of distant extremity are difficult to be motivated, they still give us a tip that the combination of different intentions might be excellent in both varieties and motivation .


Rotator cuff tears are a very common condition that is often incapacitating. Whether non-surgical or surgical, successful management of rotator cuff disease is dependent on appropriate rehabilitation. If conservative management is insufficient, surgical repair is often indicated. Postsurgical outcomes for patients having had rotator cuff repair can be quite good. A successful outcome is much dependent on surgical technique as it is on rehabilitation. Numerous rehabilitation protocols for the management of rotator cuff disease are based primarily on clinical experience and expert opinion. This article describes the different rehabilitation protocols that aim to protect the repair in the immediate postoperative period, minimize postoperative stiffness and muscle atrophy.A review of currently available literature on rehabilitation after arthroscopic rotator cuff tear repair was performed to illustrate the available evidence behind various postoperative treatment modalities.There were no statistically significant differences between a conservative and an accelerated rehabilitation protocol . Early passive range of motion (ROM) following arthroscopic cuff repair is thought to decrease postoperative stiffness and improve functionality. However, early aggressive rehabilitation may compromise repair integrity.The currently available literature did not identify any significant differences in functional outcomes and relative risks of re-tears between delayed and early motion in patients undergoing arthroscopic rotator cuff repairs. A gentle rehabilitation protocol with limits in range of motion and exercise times after arthroscopic rotator cuff repair would be better for tendon healing without taking any substantial risks. A close communication between the surgeon, the patient and the physical therapy team is important and should continue throughout the whole recovery process. Symptomatic disease affects between 4% and 32% of the patients’ population with rotator cuff tears . Althougd method . Beyond d method . As a reThe rotator cuff consists of the tendons of subscapularis, supraspinatus, infraspinatus and teres minor muscles. Its main role is to dynamically stabilize the glenohumeral joint, prevent upward migration of the humeral head, contribute to the initial phase of shoulder abduction and provide internal and external rotation , 8.via fibrocartilage /scar tissue [. The mechanical properties of this fibrous tissue are weaker than the native insertion site and may render repairs prone to failure [Understanding the biology and the biomechanical factors of the injured and, of course, of the repaired RC tendon is an essential component, ensuring a successful rehabilitation protocol. Histological studies suggest that there are three phases in RC healing process that occur immediately after surgical repair: inflammatory phase (7 days), proliferative phase (2 to 3 weeks) and maturation or remodeling phase (12 to 26 weeks) . It is ir tissue , 14. Nor failure .et al. [vs degenerative).The primary goals of postsurgical rehabilitation after RC repair are protection of the healing process and parallel prevention of joint stiffness and muscle atrophy. However, there are not many high-level evidence-based studies in the English literature regarding the postoperative rehabilitation protocols. Some authors support the traditional-conservative program, while others present that both protective and accelerated rehabilitation protocols, based on individual patient, minimize the risk for developing postoperative stiffness without compromising the final result. Ghodadra et al.  showed tThe most common complication after RC surgical repair is stiffness, regardless of an arthroscopic or open technique. Postoperative stiffness can be an undesired outcome, resulting from prolonged immobilization. In general, rehabilitation protocols need to take into account that tendon to bone healing is slow. However, animal models have shown that postoperative rehabilitation reduces the tension of rotator cuff repairs and improves the healing process. There are many discussions about the duration of immobilization. Most authors agree that a period of immobilization 4 to 6 weeks is beneficial during the early postoperative phase . After tet al. showed there is less tension on the superior rotator cuff in 30ο and 45ο compared to 0 and 15οof abduction [Another factor that is usually under discussion is the arm position during immobilization. Arm sling in abduction is now widely suggested while many studies have shown that vascularization is improved and tension of the repaired tendon is minimized in this position . Hatakeybduction .et al. [The benefits of continuous passive motion after rotator cuff repair are not clear. Researchers suggest that CPM minimizes postoperative pain and appears to be more significant when it is used in conjunction with traditional physical therapy. Many authors reported no significant difference in clinical outcome of patients treated with CPM during the first 3-4 weeks following rotator cuff repair in comparison to those treated without it. Lastayo et al.  in a levet al. .The role of cryotherapy in the early postoperative period is to decrease pain, swelling, minimize the inflammatory response and improve muscle spasm. Several studies have shown that cryotherapy decreased pain and narcotic medication requirement in the immediate post-operative 24 hours compared to the control group [The benefits and the clinical advantages of water have been utilized for centuries as a mean for rehabilitation, relaxation and training . Aquaticο abduction in the water presented less electrographic activity than land-based exercises [By utilizing a pool for rehabilitation, the patient’s goal of improving range of motion can be achieved easier and much earlier than land-based exercises without compromising the repair integrity . Gollandxercises . MoreoveAs mentioned, the ultimate goal of a rehabilitation program after rotator cuff repair is to maintain secure the tendon to bone attachment and prevent shoulder stiffness. An ideal rehabilitation program should be based on close communication between the surgeon and the physical therapy team. It is important to know all the necessary details about the surgery, such as the size of the tear, the exact tendons involved, the quality of tissue and the repair technique used, in order to establish a suitable protocol for the patient. A physical therapist should take into account that every patient and every rotator cuff tear is not the same. For example, geriatric patients or large to massive rotator cuff tears may require a slower form of rehabilitation in order to avoid the risk of re-tear. On the other hand, younger patients or smaller tears with good tissue quality are more likely to undergo an accelerated “return”. In addition, there are many risk factors for postoperative stiffness such as adhesive capsulitis, concomitant labral repair, single tendon rotator cuff repair, coexisting calcific tendinitis and diabetes. Patients with all these factors should start an earlier rehabilitation protocol to avoid postoperative stiffness . For theLastly, the physical therapist should take into consideration the clinical goals, the pre-operative activities and the needs of each patient. This entails having a lengthy discussion with the patient before and after the surgery in order to cover every aspect of patient’s recovery, to ensure his compliance with the procedure and finally his return to daily activities.One of the purposes of this review is to present an approach of common rehabilitation protocols after rotator cuff repair. In general, as mentioned above two different post-operative approaches exist: a moderate or accelerated rehabilitation program and a more conservative one. The moderate protocol will be presented in details (phases and exercises). The conservative-traditional protocol is characterized by a delay in each phase of moderate program (2 to 4 weeks) and a restriction of passive range of motion (2-4 weeks).Rotator cuff repair is followed by a program consisted of four commonly used and accepted phases, beginning with the aim to protect mainly the repair in the immediate postoperative phase and gradually return to preoperative activity level .At this phase, one of the main goals is to educate the patient and to establish a close communication in order to understand its personal desires and goals and collaborate throughout the whole recovery process. It is also important, as previously mentioned, to accumulate a detailed medical pre- and intra-operative medical history. Regardless of the determined protocol, at this initial phase, the therapist should focus on maintaining the integrity of the repair.s position may enhance regional blood flow by preventing the “wringing out” effect in blood vessels to the tendon and may reduce the distance between the origin and the insertion of the muscle tendon unit so that passive tension on the repair site will be decreased [At phase I, patients are expected to wear their arm immobilizer with the abduction pillow for 6 weeks post-operatively unless specifically directed to do otherwise by the operating surgeon . This deecreased . MoreoveIt is widely described that early passive motion may be beneficial in helping the process of tendon to bone healing. Passive ROM exercises should be performed with gentle oscillations. Pendulum exercises can also be performed by leaning the body with the support of a chair and having the arm dangle in front of the body and by performing clockwise and counter clockwise motion. At this phase, cryotherapy can be helpful regarding postoperative pain and inflammation, so the patients should be instructed on the use of ice at home. Considering the sleeping position, during the first few weeks postoperatively it is usually more comfortable for the patient to sleep in a recliner with a pillow underneath the operative extremity while wearing the abduction sling. Aqua therapy may be also beneficial in the second to sixth week. Furthermore it is equally important the proper education of the patient concerning what to avoid at home. There are many precautions at this phase such as lifting heavy objects, pushing and pulling, excessive shoulder extension, excessive movements behind the trunk stretching or sudden movements, leaning on the elbow supporting of body weight by hand with transfers in and out of bed and chair .o - 125o. 2. Passive external and internal rotation in arm immobilizer to at least 25o -45o. 3. Passive glenohumeral abduction in arm immobilizer to at least 90o.The criteria to progress from phase I to phase II is a pain free passive motion. More specific: 1. Passive forward flexion to at least 110By six weeks postoperatively the inflammatory and repair phases have progressed to the collagen remodeling stage. During this time tendon healing to bone is progressively increasing and the application of low level muscle forces aids in orienting the fibers within the collagen matrix and enhancing the tensile strength of the tendon repair . This pho of forward elevation. Then the patient is instructed to draw circles or the alphabet in the air utilizing small, controlled, motions. Moreover in this time, sub-maximal isometric external and internal rotation can be initiated. These exercises are performed holding the arm below shoulder height, elbow flexed to approximately 90o, and held in a neutral rotation position. A tower roll is placed between the elbow and trunk and the patient is asked to push into internal or external rotation, resisting with the uninvolved limp, starting at approximately 25% of maximal effort and gradually increasing to 50-70% of maximal effort without pain [Five to 7 weeks after surgery,open-chain proprioceptive exercises can be added to rehabilitation protocol. These exercises help to restore muscle strength and proprioception and are performed with the patient in supine position and the involved upper extremity held in 90out pain .During this phase aquatherapy can be more advanced adding active motion as it is considered to be active-assisted ROM in a gravity reduced environment. Studies have shown that shoulder elevation in the water is less active for the rotator cuff compared to dry land exercise .Although the progress, patient in this phase should continue avoiding resistance or strength activities. The criteria to progress to phase III are full active ROM compared to the contralateral arm and no signs of scapular-thoracic dyskinesia .Phase III can be called a strengthening phase, taking place approximately 10 to 12 weeks postoperatively. In this phase, the histological remodeling phase is complete and tendon to bone healing is strong enough to allow a strengthening program. The main goals of this phase is having a full passive ROM without pain, optimizing neuromuscular control and improving endurance. The patient must take into consideration that attempts to strengthen a stiff shoulder can cause pain and stress on the repair, so a strengthening program is permitted only when shoulder mobility and ROM are maximized.During this phase, the patient begins with stretching and strengthening exercises and continues with elastic resistance activities in order to build muscles endurance. These exercises are external rotation, internal rotation, forward flexion and rowing motion, which are performed with a towel roll placed between the arm and trunk. Free exercises can be used to strengthen the biceps and triceps muscles. Generally, patients should have regained at least 80-90% of their range of motion at this phase, unless they had a large or a massive tear requiring lateral mobilization of the tendon or a more extensive procedure. When the patient is pain-free with activities of daily living and tolerates all strengthening exercises without pain, then can continue to phase IV.o of abduction utilizing elastic resistance. Then perform external rotation exercises at 90o of abduction in order to activate supraspinatus muscle. Additionally, the push up with a plus progression from a wall to a chair and then finally to the floor is a more advanced exercise that strengthens the serratus anterior muscle. Finally, upper limb plyometric exercises, known as “jump training” or “plyos”, which for example include a patient throwing and catching a weighted ball against a wall, starting at shoulder height and progressing gradually to overhead. It is thought that these exercises improve neuromuscular control, strength, and proprioception.Phase IV is the advanced strengthening phase that typically can be initiated approximately 16-22 weeks after rotator cuff repair. At this point the remodeling phase should be complete and the repaired rotator cuff tissue is relatively mature enough to withstand greater forces. Progressive strengthening of repaired rotator cuff can be achieved in many ways. To ensure high levels of infraspinatus and teres minor strength, perform external rotation of the shoulder at 45At this phase it is also necessary to choose specific exercises that target the scapular stabilizers. An exercise that aims to strengthen the serratus anterior muscle is having the patient standing and facing away from the elastic resistance attachment with the hands held at shoulder width and chest height, holding onto the resistance band. Then the upper extremities are extended forward away from the body at 120 degrees of forward elevation. An additional exercise that strengthens the serratus anterior muscle is the push-up with a plus progression, first beginning with pressing against a wall, then progressing to the edge of a table and finally to the floorHowever the restrictions in this phase are overhead lifting and return to competitive sport. Finally, the criteria to return a patient to pro-tear conditions or to normal everyday activities are: 1. Symmetrical ROM and strength, 2. Normalized scapulothoracid kinematics, 3. No pain at rest or with activities.et al. [versus those of a traditional rehabilitation protocol. The authors included 6 studies and investigated the effects and the differences of the early passive ROM exercise. The results showed that the aggressive rehabilitation protocol was superior to the traditional protocol regarding the outcomes of overall ROM at 6 months and 1 year after repair, and finally led to greater improvement in shoulder function. However despite the better and earlier functional results, an aggressive rehabilitation protocol may entail higher risks of rotator cuff un-healing and a higher retear rate than the traditional protocol.The postoperative rehabilitation protocols regarding the duration of immobilization, passive/active motion, and aggressive post-operative treatment are still under debate. Huang et al.  in a rec1.The current literature on rehabilitation following rotator cuff repair and its postoperative effects can be summarized in the Table Rehabilitation following rotator cuff repair begins with a close communication between the surgeon, the patient and the physical therapy team. It is important that this communication should continue throughout the whole recovery process. The physical therapist should collect all the information and details that are useful in order to create an appropriate and successful rehabilitation protocol. These protocols vary between providers with respect to timing of progressing and appropriate therapeutic exercise. Either a conservative or a moderate rehabilitation protocol is selected, the main goals should always be the maintaining of the repair, the minimizing of tendon stress and pain and the faster return to the previous activities of patients life. A conservative protocol is characterized by either a delay in the initiation or/ and restriction of passive ROM while a moderate /accelerated rehabilitation program is characterized by initiation passive ROM on postoperative time. Nowadays all protocols are commonly based on clinical experience and expert opinion rather than scientific rationale.


These androgenic responses were attenuated by gonadotropin treatment in vivo. Growth of antral follicles from DHT-treated rats in vitro was significantly slower when compared to those of control but was significantly enhanced by exogenous GDF9, suggesting the DHT-induced antral follicular growth arrest is in part the results of GDF9 suppression. Our findings indicate how hyperandrogenism modulates RNF6 content and subsequently AR ubiquitination, resulting in antral follicle growth arrest in a chronically androgenized PCOS rat model.Although chronic hyperandrogenism suppresses antral follicular development, a phenomenon often observed in polycystic ovarian syndrome (PCOS), whether and how deregulation of androgen receptor (AR) signaling is involved, is not well understood. In the present study, we examined the role of ring finger protein 6 (RNF6) in AR ubiquitination and the possible dysregulation in the expression and actions of growth differentiation factor 9 (GDF9) and kit-ligand (Kitlg) in a chronic androgenized PCOS rat model. 5α-dihydrotestosterone (DHT) treatment  Polycystic ovarian syndrome (PCOS) is a heterogeneous syndrome affecting 10% of women in reproductive age and accounts for 75% of anovulatory fertility. It is associated with antral follicle growth arrest, suppressed proliferation and enhanced apoptosis of granulosa cells and hyperandrogenemia7. The molecular and cellular mechanisms involved in antral follicular growth arrest in PCOS are not well understood.Androgens stimulate granulosa cell proliferation and promote preantral follicle growth in the mammalian ovary8. Protein ubiquitination involves the binding of ubiquitin to substrates as single ubiquitin (mono-ubiquitination) or as ubiquitin chain (poly-ubiquitination). The ubiquitination process involves sequential action of ubiquitin-E1, E2 and E3, whereas E3 is a determinant of substrate specificity10. Ubiquitin contains seven lysine residues K-6, 11, 27, 29, 33, 48, and 63 through which the ubiquitination chain extends11. It is well established that polyubiquitination at K48 and K63 leads to protein degradation by 26 S proteasome12 and transcriptional activation13, respectively. Small nuclear RING  finger proteins are E3 ligases and are nuclear receptor co-regulators15. Ring finger protein 6 (RNF6), a member of this family, induces AR ubiquitination8. RNF6 is believed to promote AR transcriptional activity16 or AR proteasome degradation17 and it appears to be cell type-specific and dependent on stage of cellular differentiation. We have recently demonstrated the role of RNF6 in AR signaling in the regulation of granulosa cell fate during preantral follicle growth. RNF6 is a positive mediator in androgen-induced AR polyubiquitination, Kitlg mRNA expression and granulosa cell proliferation in vitro18. However, whether and how RNF6 is important in the dysregulation of follicular growth in PCOS is not known.Androgen receptor (AR) plays important regulatory roles in ovarian follicular development. It is well established that the cellular actions of androgens are mediated via its binding to and activation of AR, which regulates gene transcription in androgen-dependent cell growth and proliferation. AR signaling is regulated by a variety of posttranslational modifications, including ubiquitination, phosphorylation, acetylation, methylation and sumoylation20 and the oocyte-derived factor growth differentiation factor 9 (GDF9)21. Depending on the stage of follicular development, androgens could promote or suppress ovarian follicle growth23. While androgens enhance preantral follicle growth by stimulating the GDF925, chronic androgenic stimulation induces antral follicle growth arrest and polycystic ovarian syndrome phenotypes in the rats by GDF9 down-regulation26. Kitlg signals through its receptor Kit in the oocyte and stimulates oocyte growth and folliculogenesis. Androgen is known to up-regulate granulosa cell expression of Kitlg and its action in the ovarian follicle1. Although GDF9 and Kitlg are known to mediate androgen action on follicular development, the cellular mechanisms involved in the AR ubiquitination induced by androgen are not completely understood.Follicular cell proliferation and survival are tightly regulated by follicular factors including granulosa cell-derived factor KitlgKitlg mRNA expression) and AR stability and abundance (K48). During chronic androgenization, increased RNF6-mediated AR ubiquitination (K48) results in enhanced AR degradation and decreased Kitlg expression, GDF9 down-regulation and antral follicular growth arrest. Our studies suggest that antral follicle growth arrest observed in PCOS may in part be associated with androgen-induced, RNF6-mediated AR (K48) polyubiquitination and degradation and loss of granulosa cell proliferative response.The overall objective of the present studies was to examine the androgenic regulation of RNF6 expression and the role of RNF6-mediated AR ubiquitination in the dysregulation of ovarian follicular development, using a chronically androgenized rat PCOS model. In addition, whether and how these processes alter the expression of GDF9 and Kitlg was determined. We hypothesized that androgen-induced ovarian follicular growth is dependent on site-specific RNF6-mediated AR polyubiquitination, which determines AR transcriptional activity . Bovine insulin, human transferrin, ascorbic acid, sodium selenite anhydrous, L-glutamine, sodium pyruvate, agarose (low gelling temperature), HEPES, equine chorionic gonadotropin (eCG) were purchased from Sigma  and 5α-dihydrotestosterone (DHT) was purchased from Steraloids . Primary and secondary antibodies used in the present studies are shown in Supplementary Table Female Sprague Dawley rats were obtained from Charles River Canada  and maintained under standard conditions. All animal procedures were carried out in accordance with the guidelines of the Canadian Council on Animal Care and approved by the University of Ottawa Animal Care Committee. Briefly, 21 days-old rats were divided into two groups  and implanted subcutaneous for 30 days with SILASTIC brand capsules  containing 7.5 mg DHT (continuous daily release: 83 µg). Control rats received identical capsules without DHT. Granulosa cells were isolated from “preantral follicles”  and “antral follicles” (150–200 µm) for protein and mRNA analyses.27. Depending on the design of the experiments, hormonal treatments were initiated at the beginning of culture. Follicular diameter was measured daily as the average distance between the outer edges of the basement membrane in 2 perpendicular planes and results were expressed as the change in follicular volume. Follicular volume was calculated according to the formula for the volume of a sphere: volume = 4πr3/3, where r is radius. The percent change in follicular volume on day n of culture is defined as the volume difference between day n and day 0 (the day of follicle isolation) expressed as a percentage of the volume at day 0. The culture medium was changed every other day.Antral follicles  from CTL or DHT implanted rats were isolated in Leibowitz L-15 medium with BSA , using 28.5-gauge needles  under a stereomicroscope. Follicles with intact basement membranes and theca layers were individually cultured as described previously28. Briefly, collected follicles were lysed and protein content was determined. Equal quantities of protein lysate (40 µg) were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated  in Tris-buffered saline 0.1% tween 20 (TBST) and non-fat dry milk (5%). The membranes were then incubated  with Rabbit anti-rat AR antibody (1:5000 dilution), GAPDH antibody (1:8000 dilution), rabbit anti-rat RNF6 antibody (1:5000 dilution), mouse anti-rat Kitlg antibody (1:5000 dilution) and then with Fluor-680 labeled secondary antibodies . After washing three times with TBST, immunoreactive bands were visualized with the Odyssey infrared imaging system  at a wavelength of 700 nm. The protein signals were densitometrically scanned and quantified, using AlphaEaseFC  and normalized with GAPDH expression.Procedures for protein extraction and Western blotting have been described previously29. Isolated follicles (~300) were harvested and lysed in a buffer . In immunoprecipitation (IP) studies, cell lysates were pre-cleared (1 h) with protein A/G PLUS-agarose and supernatants were incubated with AR antibody  and treated with protein A/G PLUS-agarose . Immunoprecipitates were washed three times with 1 mL TBS. After heating , co-immunoprecipitates (K48 or K63 ubiquitin) were detected by Western blot.Cellular fractionation was performed as described previously28 and total RNA was extracted with the RNeasy mini kit, according to the manufacturer’s instructions. Extracted RNA (200 ng) was reverse transcribed into cDNA with the Sensiscript RT kit. Real-time quantitative PCR analysis was performed as previously described30. The primers which were designed to detect sKitlg (NM_021843.4), but not mKitlg used for SYBR Green real-time quantitative PCR analysis are shown in the Supplementary Table -∆∆CT method, presented as the fold change in mRNA abundance normalized against the GAPDH gene and expressed relative to the respective control.Granulosa cells were released from preantral and antral follicles by follicular puncture with a 26.5-gauge needle31. After 4% paraformaldehyde (PFA) fixation, ovarian tissues embedded in paraffin and cut into 5 µm thick sections were washed three times in PBS. Slides were blocked with 3% BSA and incubated  with primary antibodies against rabbit anti-rat AR monoclonal antibody (1:100 dilution) or rabbit anti-rat RNF6 polyclonal antibody (1:100 dilution) or mouse anti-rat Kit ligand monoclonal antibody (1:100 dilution) or goat anti-human GDF9 polyclonal antibody (1:100 dilution). After washing three times with PBS, slides were incubated  with Alexa Fluor 488 or 594 fluorescence-conjugated secondary antibodies (1:200 dilution) and mounted on slides with ProLong gold anti-fade reagent with DAPI) and optical images were obtained using a fluorescence microscope .IF were performed as described previously with modificationspost-hoc test. Significant difference was inferred at p < 0.05.All data were analyzed using GraphPad Prism 5.0 statistical software . Results are expressed as mean ± SEM of at least three independent experiments as detailed in the figures. One-way or two- ANOVA were used to assess the effects and interactions of one and two variables and multiple comparisons were achieved by a Bonferroni in vivo , a marked reduction in overall ovarian weight and size was observed in the DHT-group . There were significantly less large antral follicles  and no corpus lutea. In addition, the number of condensed atypical follicles containing primarily granulosa cells26 was significantly increased in the DHT-treated rats . Similarly, AR contents in the antral follicles decreased markedly following DHT treatment  and Western blot (WB). Both AR and RNF6 were primarily localized in the granulosa cell layer Fig . The flu16. Recently, we have demonstrated that DHT increases AR polyubiquitination in granulosa cells from immature follicles at lysine residue 63 but not at lysine residue 4818. Mono-ubiquitination of AR at K63 and K48, often associated with intracellular transport, was not significantly affected by DHT. Androgen markedly increased K63-linked AR poly-ubiquitination in granulosa cells and this response was significantly inhibited by RNF6 silencing in vitro18. To determine whether chronic androgen treatment in vivo influence follicular development through its RNF6-mediated AR actions, we compared K63- and K48- AR poly-ubiquitination by co-precipitation at pre-antral and late antral follicular stage without/with DHT treatment. AR in antral follicles from DHT-treated rats exhibited a marked decrease in K63-linked poly-ubiquitination  but not of preantral (P > 0.05) follicles was markedly down-regulated in DHT-treated rats when compared to control rats. To examine whether antral follicular growth arrest in DHT-treated rats is indeed related androgen-induced GDF9 down-regulation, antral follicles from DHT-treated or control rats were cultured for 4 days in the absence and presence of GDF9 (100 ng/ mL), and the growth was assessed. Antral follicles cultured in the absence of GDF9 exhibited minimal growth (Fig. P < 0.01 CTL + GDF9 vs. CTL; P < 0.001 DHT + GDF9 vs. DHT Fig. in vitro.GDF9 is expressed in the mammalian oocyte throughout folliculogenesis and stimulates proliferation and suppresses apoptosis in granulosa cellswth Fig. . Howeverin vivo. Androgenic stimulation resulted in marked increase in RNF6 content and down-regulation in AR levels in antral follicles. Those two responses closely associated with suppressed AR K63-linked poly-ubiquitination and enhanced AR K48-linked poly-ubiquitination. Additionally, we also have demonstrated that DHT effects were attenuated by gonadotropin treatment in vivo. Our studies also support the notion that androgen suppresses antral follicle growth in part by inhibition of sKitlg and GDF9 expression, since antral follicles growth in the DHT-treated rats could be restored by exogenous GDF9 in vitro.Using a chronically androgenized rat model, we have investigated in the present study the role of RNF6-mediated site-specific AR ubiquitination in androgen-induced antral follicular growth arrest. We have demonstrated that chronic DHT treatment inhibited antral follicle growth during folliculogenesis in the rat 37. It is often manifested with dysregulated folliculogenesis and reduced fecundity38. Chronic and abnormally high androgen levels suppress antral follicular development39. It has been previously demonstrated that androgen resulted in suppressed antral follicular growth and estrogen secretion in the rats and mice41. In addition, testosterone or DHT supplementation in pig oocytes cultures medium significantly increased the proportion of oocytes arrested in the metaphase I stage42. Suppressed ovarian follicular maturation has been reported after neonatal androgen administration43, as also evident in PCOS39. Our current studies clearly show that DHT significantly induces follicular growth arrest in the antral follicle stage but not in the preantral follicles in vivo. It suggests that the role of androgen in the regulation of ovarian folliculogenesis is follicle stage-dependent. Specifically, androgen is a negative effector during the antral follicle development.PCOS is commonly associated with anovulatory fertility. Features of this syndrome also include hyperandrogenism, insulin resistance, immune disorder and multiple hormone imbalances16, there is no report on whether RNF6 modulates proteasome degradation of AR or enhances AR transcriptional activity in an androgen-induced PCOS rat model. Our current studies show that DHT increases K48-linked poly-ubiquitination of AR and decreases K63-linked poly-ubiquitination of AR in antral follicles in vivo. It suggests that RNF6 plays an important role in androgen-induced AR degradation during antral follicle growth in vivo.Although biochemical and mass spectrometry analyses suggest direct interactions between the E3 ligase RNF6 and AR21 and that its expression is regulated by androgen31. In a rat PCOS model, chronic DHT treatment inhibits GDF9 expression and antral follicle growth26. Although mRNA abundance of GDF9, but not BMP15, in human oocytes has been reported to be decreased in PCOS44, whether antral follicle arrest in PCOS is due to androgen-induced, RNF6-mediated AR ubiquitination and AR down-regulation and subsequent dysregulated GDF9 expression is not known. We have previously demonstrated that androgen up-regulates RNF6, AR and granulosa cell proliferation in pre-antral follicles in vitro18 and that RNF6 knockdown or androgen receptor antagonist treatment inhibits the growth of these follicles45. In the present studies, we have demonstrated that DHT increased site-specific AR polyubiquitination, AR degradation and decreased GDF9 expression. Moreover, the androgen-induced antral follicle arrest in vivo appears in part due to down-regulation of GDF9 expression, since this response could be attenuated by exogenous GDF9 in vitro. Taken together, antral follicle growth arrest observed in PCOS may be due to a loss in androgen responsiveness resulting from down-regulation of AR following K48 polyubiquitination and of androgen-induced GDF9 expression.GDF9 is important promoter of preantral follicular growthKitlg expression and granulosa cell proliferation in preantral follicles18. Although GDF9 and Kitlg are known to mediate androgen action on follicular development, the cellular mechanisms involved in the AR ubiquitination are not completely understood. In the rat, Kitlg is expressed in granulosa cells47 and its receptor in the oocyte and theca internal cells48. These findings raise the interesting possibility that androgen up-regulates sKitlg mRNA expression and protein content, and therefore indirectly promotes preantral follicular growth through the expression and action of oocyte-derived GDF949. The dysregulation of these regulatory mechanisms may be important in the ovarian pathology of PCOS. Our observation in the present study that DHT suppresses the expression of sKitlg and GDF9, and antral follicle growth in vivo is consistent with this notion. In contrast, Tuck et al., reported that granulosa cells of human subjects with PCOS had a greater intensely of KIT-L than normal subjects. These discrepancies may be associated with differences of human PCOS compared to animal models which need to be further assessed50.We have previously demonstrated that RNF6 is a novel positive mediator in androgen-induced site-specific AR polyubiquitination and AR transcriptional activity for s39. It is used commonly in infertility therapy including controlled ovarian stimulation in in vitro fertilization and for PCOS patients45. It offers a safe and successful treatment option in patients with PCOS with an acceptable risk for multiple gestations51. Our previous and present studies show that gonadotropin enhances sKitlg mRNA and protein contents on the antral follicles and attenuates DHT-induced antral follicles arrest in vivo34. Therefore, defining the action and interaction of gonadotropin and GDF9 could provide important clue for treatment of dysregulated follicular development in PCOS.Gonadotropins stimulate the growth and recruitment of ovarian follicles in the ovary. In early antral follicles, FSH is a cell survival factor that rescues the small antral follicles from apoptosisin vitro in a follicle stage-dependent manner by inducing RNF6-mediated, site-specific AR polyubiquitination and altered AR transcriptional activity and stability45. Using rat model for PCOS, we have demonstrated that androgen induces antral follicle growth arrest through AR polyubiquitination and down-regulation. To facilitate future investigation on the role of RNF6 in the regulation of folliculogenesis and its deregulation in PCOS, the following hypothetical model is proposed (Fig. via increased RNF6-mediated K48 site-specific AR poly-ubiquitination and increased AR proteasome degradation. Dysregulation of this pathway suppresses sKITLG and GDF9 expression, leading to growth arrest of the antral follicle. Gonadotropin stimulation in vivo restores follicular growth. Although these findings are intriguing, their pathologic significance in human PCOS remains to be explored. Our findings significantly contribute to the current understanding of the role of RNF6 in AR signaling in the control of ovarian follicular growth and its possible dysregulation in PCOS. It provides novel insights in the pathophysiology of PCOS associated patients with androgen excess.We have previously shown that androgen regulates ovarian follicular growth Supplementary Information


Allergic rhinitis (AR) is associated with various developmental issues that affecting dentition. We aimed to determine whether AR is associated with an increased risk of traumatic dental injuries (TDIs) in Taiwanese individuals. We used the Taiwan National Health Insurance Research Database (NHIRD) to conduct a nested case-control study. We compared an AR cohort with a matched cohort of patients without AR. New TDI cases were determined during our study period. To compare TDI risk between our study cohorts, we used Cox proportional regression analysis, and hazard ratios (HR) with 95% confidence intervals (CI) were calculated to quantify the association between AR exposure and TDI risk. In total, 76749 patients with AR  were identified. In the AR and the non-AR cohorts, 312 patients in total had TDI. Patients with AR had a significantly higher risk of TDI than those without AR . The risk of TDI was markedly higher in the AR cohort, except in the 3–12-year-old group, and with a CCI ≥ 1. AR patients had a future risk of TDI, indicating a potentially linked disease pathophysiology. The association between AR and TDI is greater among general patients. Clinicians and caregivers should be aware of potential TDI co-morbidity in patients with AR. Allergic rhinitis (AR) is the most common airway-obstructing disease, affecting 17.9%–26.3% of adolescents . AdditioThis raises the question of whether AR may lead to traumatic dental injury (TDI). TDI is a serious dental public health problem that results in fractured, displaced, or lost teeth and can have significant negative functional, esthetic, and psychological effects. In addition to pain and increased infection risk, the consequences of TDI include changes in physical appearance and speech defects, and can thus affect the patient’s quality of life. TDI is more time-consuming and costly to treat than many other outpatient accidental injuries .TDI occurs frequently in children and young adults, comprising 5% of all injuries. Boys are generally injured more frequently than girls . GeneralOur study aimed to determine the risk of TDI in patients with AR in Taiwan. We conducted a retrospective cohort study to estimate the relative risk of the incidence of TDI in a nationwide AR cohort, as compared with a non-AR cohort selected from a 1 million representative population. We hypothesized that we would be able to predict the tendency for TDI in AR patients. In the high-risk group, more attention was given to prevention of TDI by preventative and interceptive orthodontic treatment and by providing a safe environment. Furthermore, these findings may be used to influence public policies in long-term care and childcare.This study employed a retrospective cohort study design.The Taiwan National Health Insurance Program was established in 1995. This system provides universal health coverage and equal medical access to all Taiwanese individuals. In 2011, the coverage rate of the National Health Insurance was 99.6%; almost the entire population of Taiwan (23 million) was enrolled in this program. A computerized database  was set up, derived from the Taiwan National Health Insurance Program and managed by the Taiwan National Health Research Institute (NHRI), which is a nonprofit foundation established by the government. The NHIRD includes patients’ demographic information, encrypted identification numbers, sex, birth dates, admission dates, diagnostic data and procedures, dates of diagnosis, dates of medical treatment, International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnostic codes, and drug codes.We conducted a nested case-control study using the NHIRD. From 2000 to 2008, nationwide cohorts were identified on the basis of diagnostic codes: AR and non-AR codes. The cohorts included in our study included individuals aged > 3 years.The NHIRD encrypts patients’ personal information to protect privacy, and provided researchers with anonymous identification numbers associated with relevant claims information, including the patient’s sex, date of birth, medical services received, and prescriptions. Therefore, patient consent was not required to access the NHIRD .http://nhird.nhri.org.tw/).All data and related metadata were deposited in an appropriate public repository. The data on the study population obtained from the NHIRD are maintained in the NHIRD  in 2000, whereas those diagnosed with AR before that year were excluded. In addition, we excluded patients with any acute conditions, such as those with ICD-9 codes for physician-diagnosed cerebrovascular disease and epilepsy, to reduce possible confounding effects.For comparison, the non-AR cohort was identified from among individuals aged > 3 years and who had no AR diagnostic codes. Each patient with AR was matched to a non-AR patient on the basis of age, sex, duration of enrollment, and cohort entry date. All patient in our cohorts were followed up until dental trauma occurred or until 2010.As outcomes, we focused on the risk of dental trauma with AR. During our study period, we determined all physician-diagnosed dental trauma cases. Diagnostic codes for dental trauma included ICD-9:873.63—open wound of tooth (broken) (fractured) (owing to trauma), without mention of complications—and 873.73—open wound of tooth , complicated.Chi-square tests were used to compare the baseline characteristics between our two cohorts. We used Cox proportional regression models to compare the risk of dental trauma and to determine adjusted hazard ratios (aHR), which were adjusted for patients’ age, sex, and their Charlson comorbidity index (CCI) score. The non-AR data were used as a reference. A P-value of < 0.05 was considered statistically significant. The cumulative incidence curves of dental trauma in the study cohort were estimated using Kaplan-Meier analyses and the differences between the two cohorts were compared using the log-rank test. All analyses were performed using SAS software, version 9.2 .In total, 76749 patients with AR  were identified during the period of 2000–2008, as shown in There were no significant differences between the AR and non-AR cohorts in terms of age, sex, and duration of follow-up, as shown in During our study period, 312 dental trauma cases were identified in the cohorts, involving 218 patients with AR and 94 without AR, as shown in TDI is a condition with a poor prognosis that influences appearance and function. Our aim was to estimate the risk of TDI in patients with AR, to determine the need for further preventive treatment and for measures to make the environment safe. The database used in this study was released for research purposes by the National Health Research Institutes in 2008. As of 2007, 98.4% of Taiwan’s population (approximately 22.96 million) was enrolled in the NHIRD. The data of the present study were retrieved from 1 million randomly sampled enrollees from the total NHIRD. In this nationwide, population-based, randomized, and longitudinal study, we demonstrated an increased risk of TDI in AR patients, which was 1.92-fold higher than that in the non-AR cohort, after adjustment for sex, age, and medical comorbidities. In each group, the risk of TDI was markedly higher in the AR cohort, except in the 3–12-year-old group, and with a CCI ≥ 1. In the CCI (0) group in particular, excluding most systemic diseases would be more near really risk of TDI with AR. These results support our hypothesis that patients with AR have a higher prevalence of TDI,Airway obstruction is the main syndrome of AR, even if it is not a continuous phenomenon. AR is considered to be one of the major causes of airway obstruction in children , which cIn our study, we excluded patients with cerebrovascular disease and epilepsy to avoid the factors of iatrogenic injury and illness. However, the risk of TDI may be affected by the presence of other systemic diseases that cause oral complications, such as periodontitis or trismus. Deyo’s CCI score differed significantly between the AR and non-AR cohorts, with the AR cohort having more systemic diseases. This tendency may elevate the risk of TDI. To identify the health levels in patients with AR, we used the CCI to discriminate between patients with AR and those without AR, in 3 groups: low (CCI = 0), moderate (CCI = 1) and high (CCI ≥ 2). We found statistically significant differences between cohorts in the low CCI group , but not in the moderate and high CCI groups. The results expressed the risk of TDI between AR and non-AR patients affected by systemic diseases; the CCI = 0 group approximated the risk of TDI between AR and non-AR patients without systemic diseases.In the various age groups, the primary and mixed dentition group did not attain statistically significant differences between the groups . In the AR also affects human behavioral disorders, such as ADHD and oppositional defiant disorder, particularly among children . NeverthThe study had the following limitations. First, the NHIRD did not include orthodontic and cosmetic patients; such corrections may reduce the developmental disorder caused by AR. Second, the maximal follow-up time of our study was 8 years; however, developmental disorders caused by AR persist life-long. Although there were clear differences in the follow-up time between patients with AR and those without AR , furtherIn conclusion, this large-scale, nationwide, population-based, longitudinal study demonstrated an association between AR and the risk of TDI. Although the exact mechanism is presently unclear, these findings may provide further insights into the association and possible shared pathophysiology between AR and TDI. The importance of prevention measures for TDI in patients with AR should be emphasized by ensuring a safer environment. Furthermore, these findings may be used to influence public policies in long-term care and childcare.S1 File(PDF)Click here for additional data file.