A New Hindlimb Model for Inducing Extensor Digitorum Longus Atrophy in Rats

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A New Hindlimb Model for Inducing Extensor Digitorum Longus Atrophy in Rats

    A New Hindlimb Model for Inducing Extensor Digitorum Longus

    Atrophy in Rats? Tail suspension-immobil


     0 Introduction

     Available literature suggests that skeletal muscles, especially the antigravity muscles (mainlythe slow twitch muscles), are prone to disuse atrophy [1-5] . These muscles show reduction of massand the cross sectional area, (CSA) within 10-day-inactivity, bed rest or spaceflight(weightlessness). In addition, a shift from slow muscle fibre (mainly the type I fibre, e.g. soleus) tofast muscle fibre (mainly the type II fibre, e.g. extensor digitorum longus, EDL) also occursfollowing deconditioning induced by these conditions [6, 7].

     Tail suspension and immobilization are two common hindlimb disuse atrophy models. Bothlead to disuse atrophy of the soleus and the EDL [8-11] and in soleus, a morphological transitionfrom type I fibres to type II fibres occurs [12-15]. However, the effects of these two models on thefibre transitions of the EDL [8, 11, 16, 17] are not consistent. For instance, a study reported decreasesin type IIB fibre proportion of EDL after 3-week-tail suspension [16] but another one reported adecreased percentage of type I fibre of EDL after 5-week-tail suspension [9]. The differences inEDL results reported between these studies may be attributed to the fact that under the conditionof tail suspension animals instinctively contract their EDL for support. Therefore, tail suspensionmodel may not simulate the effects of disuse (like bed rest and spaceflight) on the EDL to somedegree. By contrast, the plaster casting (immobilization) should overcome the above adverse effect.However, when the animal pulls it?s immobilized limb, it will contract instinctively it’s EDL forsupport, which will also weaken the disuse effect.

     The present study was aimed at establishing a new hindlimb disuse atrophy model. This studyexamined the effects of a tail

    suspension-immobilization (TS+Im) and tail suspension only (TS)model on the morphous and fibre type distribution of the EDL of rats. The results of the study areimportant as a model, which leads to quicker onset of disuse atrophy would reduce the amount oftime the animals have to be subjected to tail suspension.

     1 Methods

     1.1 Animals and Grouping

     48 adult female Sprague-Dawley (SD) rats were randomly divided into 8 groups matchingtheir body weight ((200?20) g): 1 week (wk), 2wk, 3wk, 4wk, 5wk, 6wk and 7wk of modeling andthe control group. Each group had 6 rats. Each rat was reared in a single cage, measuring 30 cmwide, 35 cm deep, and 45 cm in height.

     1.2 Hindlimb Unloading Animal Model

     The hindlimb unloading model was established according to previously describedtail-suspension method [18]. An orthopedic tape-adhesive plaster, covering less than half of thecleaned and dried tail, was linked by a shoelace to the top of the cage; a swivel joint allowed 360 rotation. The length of the lace was adjusted to incline the body at 30? from the horizontal plane,but the animals could move freely on the forelimbs. One hind limb of the rat was kept suspended(tail suspension limb only, TS limb); the other hind limb was immobilized by plaster bandagewinding from 1 cm above the ankle to the toe, and this is the tail suspension plus immobilizationlimb (TS+Im limb) (Figure 1).

     1.3 Sample Preparation and Histochemical Staining

     Animals were anaesthetized with 45-mg/kg intraperitoneal sodium pentobarbital; the EDLmuscle was then removed. 3 mm-long muscle samples were cut from the mid-belly of the EDLand put into a pre-cooled 30% saccharose solution, embedded vertically with an Optimal

    CuttingTemperature gum (Tissue-Tek?, USA).

     Using the Dubowitz-Brooke method (1973) [19], muscle sections were put into an acidpre-incubation solution (10 ml 0.2 M acetate buffer, pH4.6) for 5 min; then into a basicpre-incubation solution (2 ml 0.1 M barbital sodium solution, 2 ml 0.18 M Calcium chloridesolution, 6 ml double distilled water, pH 10.0) for 30 s. The sections were then incubated in asolution containing 2 ml 0.1 M barbital sodium, 1 ml 0.18 M Calcium chloride solution, 30 mgATP disodium, 6 mg 2,4-dinitrophenol, 7 ml double distilled water for 45 min at pH 9.4 and 37?C.

     The sections were then washed with 1% CaCl2 for 3 min three times and with 2% CoCl2 for 3 min.Finally, they underwent coloration using 1% (NH4)2S for 1min; were washed, dehydrated, clearedand mounted with gum.

     1.4 Data Acquisition and Statistical Analysis

     The type I, IIA and IIB muscle fibres were classified based on the intensity of mATPasestaining, using a light microscope[20, 21]. The CSA of single muscle fibre was measured usingMotic Images Plus 2.0. The fibre-type distribution is expressed as a ratio between the number offibres of each type and the total number of fibres. LSD was used for the interclass analysis aftersingle factor-ANOVA, analysis using SPSS

    13.0. Results of the TS limb and the TS+Im limbmodels were compared using a paired-t test. All the data (in percentage) were arcsinelytransformed; the significance level was set as p=0.05.

     2 Results

     2.1 Changes of CSA of the TS Limb’s EDL

     Following 1-6 weeks of TS, EDL CSA showed fibre dependent changes. As compared to thecontrol group, significant (p < 0.05) decreases were observed after 6 weeks in type I and type IIBfibres (-18.01% and -20.05%, respectively) (Figure 2).

     2.2 Changes of CSA of the TS+Im Limb’s EDL

     Following 1-6 weeks of TS+Im, EDL fibre dependent changes in the CSA were also seen. Ascompared to the control group, significant (p < 0.05) decreases were observed after 4 weeks intype I and type IIB fibres (-14.80% and -14.06%, respectively). CSA of type IIA fibres showedsignificant reduction after 5 weeks (-12.83%, p < 0.05) (Figure 3).Compared with the TS limb, CSA of type I fibres of the TS+Im limb’s EDL showed adecrease

    trend after 4 weeks (p =0.080). The CSA of type IIA fibres diminished significantly after5weeks (p<0.05) and greater decreases were observed after 6 weeks (p<0.01). The CSA of typeIIB fibres diminished sharply after 4 weeks (p<0.05) and showed further decreases after 5

    weeks(p<0.001)(Table 1).

     2.3 Fibre Composition of the TS Limb’s EDL

     Following 1-6 weeks of TS, fibre composition of EDL changed. The proportion of type IIAfibres and I increased gradually but type IIB fibres decreased. The most significant change of allthe three-fibres proportion was observed after 4weeks (p<0.05) (Figure 4).

     2.4 Fibre Composition of the TS+Im Limb’s EDL

     During 1-6 weeks, fibre composition of TS+Im limb’s EDL also changed. The proportion oftype I fibres decreased significantly by the 4th week (-22.59%, p<0.05). The proportion of type IIAfibres decreased significantly by 6 weeks (-30.25 %, p<0.001). The proportion of type IIB fibres,on the other hand, increased significantly after 5 weeks (+ 8.48 %, p<0.05) (Figure 5).

     3 Discussion

     Both the tail suspended limb and the tail suspendedand immobilized limb models resulted ingradual changes in the extensor digitorum longus fibre types and the cross sectional area. Both ofthese models have greater

    impact on the type I and IIB fibres but less on IIA fibre types. Inaddition, our data suggest that the effects on the muscle fibres are positively correlated with themodeling duration. As compared to Ts only, TS+Im has a faster and more pronounced effect onthe CSA of the EDL. For instance, TS+Im application resulted in decreases in the CSA of type Iand type IIB fibres (-14.80%, -14.06%) after 4 weeks and in the CSA of type IIA fibres (-12.83%)after 5 weeks. In contrast, the CSA of type I fibres (-18.01%) and type IIB fibres (-20.05%) of TSlimb’s EDL decreased only after 6 weeks; type IIA fibres CSA, however, showed no significantdecreases. These results, which are comparable with previous reports [9, 22], suggest that TS doesnot affect the CSA of the type II muscle fibres. One explanation of the different effect of the twomodels is that the TS+Im possibly prevented the active contraction of the EDL (which occursduring TS) and thus represents a true disuse atrophy model. It appears that immobilization, whileanimals are suspended, simulates the effects of disuse atrophy on the cross sectional areas of themuscle fibres more closely.

     The EDL is a typical fast-twitch muscle; it is mainly composed of type II fibres, with twosubtypes: IIA and IIB. Type I and II fibres use oxidative metabolism and glycolytic metabolism,respectively. The subtype IIA uses mixed oxidative and glycolytic metabolism; IIB, on the otherhand, relies on glycolytic metabolism. Our results show that TS and TS+Im affect muscle fibretypes of the extensor digitorum longus differently. In the TS limb, type I fibres increased while thetype IIB fibres decreased, which are in accordance to findings of Guillot C (2008)[16]. They alsoobserved decreases in type IIB fibres of the EDL in rats after 3-week- tail suspension. UsingTS+Im limb, we observed decreases in type I and type IIA fibres but increases in type IIB fibres;in other words, the muscle fibres shifted from oxidative metabolism to glycolytic metabolism[11],which is consistent and similar with the change of antigravity muscles in disuse condition.

     As both tail suspension and tail suspension plus immobilization are hindlimbdisuse-atrophy models, why do their effects on the fibre type transition differ? Wespeculate that under conditions of tail suspension, the animals retract their extensordigitorum longus muscle (perhaps by instincts, for support?), resulting in not fullimmobilization of the muscle. That may explain why the shift of muscle fibre types isdifferent from those observed in the antigravity muscles. Some reports, however, haveattributed increases of type I fibres and decrease of type IIB fibres to differences intraining[7, 9, 14]. We believe that the contradictory reports on the effects of tailsuspension on the EDL may be due to the fact that the muscle was not trulyimmobilized during testing. TS+Im isolates EDL’s movements and truly simulates completedisuse condition; it thereby induces shift in the muscle fibres as those of the antigravity muscles.

     Thus, TS+Im appears to be a better model of studying disuse effects on EDL than TS.

     4 Conclusion

     In conclusion, tail suspension with immobilization is more effective than tails suspensionalone in causing muscle fibre type changes or cross sectional area of the extensior digitorumlongus. Therefore, TS+Im can more effectively simulate the disuse atrophy of the EDL than TSonly. The TS+Im model may be superior for investigations of the morphous and the metabolicchanges, which occur in the EDL under disuse.



     [1] ADAMS G R, CAIOZZO V J, BALDWIN K M. Skeletal muscle unweighting: spaceflight and ground-basedmodels. [J]. J Appl Physiol, 2003, 95 (6): 2185?2201?

     [2] BLOTTNER D, SERRADJ N, SALANOVA M, et al. Morphological, physiological and behaviouralevaluation of a ‘Mice in Space’ housing system[J]. Journal of Comparative Physiology B: Biochemical,Systemic, and Environmental Physiology, 2009, 179 (4): 519?533?

     [3] HARGENS A R, RICHARDSON S. Cardiovascular adaptations, fluid shifts, and countermeasures related tospace flight. [J]. Respir Physiol Neurobiol, 2009, 169 Suppl1: S30?S33?

     [4] WILLIAMS D, KUIPERS A, MUKAI C, et al. Acclimation during space flight: effects on humanphysiology [J]. CMAJ, 2009, 180 (13) : 1317?


     [5] SALANOVA M, SCHIFFL G, P?TTMANN B, et al. Molecular biomarkers onitoring human skeletalmuscle fibres and microvasculature following long-term bed rest with and without countermeasures [J]. Journal of

    Analyze Jane Austen,A Transitional Writer in the English



     [ ]简?奥斯汀是英国文学史上18世纪末新古典主义过渡到19世纪初




     As we all know, it is not until the second half of the 18th century that women novelists began to appear in England. And some gifted women from the end of the 18th century to 19th century made some contributions to the development of the English novel, which even won their places in the front ranks of some realists like Charles Dickens, William Makepeace Thackeray, etc. Jane Austen (1775-1817) was just such a great woman writer in this period. She brought the English novel to its maturity and her satirical fictions marked the transition from the 18th-century neoclassicism to the 19th-century romanticism in the English literature.

     Jane Austen was born at Steventon and the seventh child of a country clergyman family. She was educated at home and passed all her life in doing small domestic duties in the countryside. She wrote six complete famous novels, including Sense and Sensibility (1811), Pride and Prejudice (1813), Mansfield Park (1814), Emma (1816), Northanger Abbey (1818), and Persuasion (1818).

     She lived and worked at the turn of the century, which witnessed a great change of the English society. Since the Industrial Revolution in the second half of the 18th century, the English social structure underwent rapid changes. The industrial capitalists began to control not only the economic but also the political power and thus the struggle between the workers and capitalists also became more and more sharp. Consequently, people started to doubt and even lose hope to the enlightening thoughts that was popular in the first half of the 18th century. Literature, as a “barometer” of the social life, also experienced great change. One of the most remarkable changes was in the literary taste. That is to say, people started to dislike Swift’s brutality, Defoe’s frank realism, and even Fielding’s masculine satire. So the works with the rational tone began to give way to the works full of sentiments.

     Under such a social and literary environment, Jane Austen exerted her transitional role in English literature. We can see it from two asp ects.

     Firstly, in theme, most of Jane Austen’s novels reflect the process of the change of the attitude towards the marriage. As we all know, in Austen’s novel Sense and Sensibility, one of the heroine, Elinor Dashwood, is quite sensible. She thinks very highly of a man whose worthiness in her eyes only increases when she learns why he cannot marry her. However, another heroine, Marianne Dashwood, is very sentimental at the beginning.

    So she trusts her senses and falls in love with a man who in truth is not as good as he seems and finally is abandoned. Through the different fates of these two sisters,Jane Austen shows us her early attitude towards the marriage that to marry according to sense will end in failure while with ration will succeed. Such a theme reflects that at first Jane Austen inherits the traditional enlightening thoughts. However, her minds change in her later novels, especially in her novels Pride and Prejudice and Persuasion. In these novels, sentiment becomes upper hand and attains success in the end. For example, at the very beginning of the novel Pride and Prejudice, Jane Austen wrote, “It is a truth universally acknowledged, that a single man in possession of a good And the hero and theroine are the very1fortune must be in want of a wife.” sensible people. Mr. Darcy, from a noble family, has a strong sense of family status and turns up his nose at the vulgarity of Bennet family. And Elizabeth Bennet is also very sensitive to her family background and becomes instantly prejudiced against Mr. Darcy, despite his good looks and great wealth. However, with the gradual deep intercourse and understanding between these two people, they fall in love with each other and finally smash the bonds of the ration and social pressure and marry. So this novel shows the victory of the sentiment, for it is just thanks to the deep love that makes these two people from different class status successfully marry. This reflects that Jane Austen’s creating thoughts begins to change from the rationalism

    to the romanticism. Her last novel Persuasion also displays such a change. Therefore, we can say that it is just through the conflict between the ration and sentiment in the novels that Jane Austen accomplishes the transition from the rational literature to the romantic literature.

     Moreover, her development from rationalism to the romanticism also reflects in her new concept of marriage. That is she advocates the only base of marriage should be the true love, not the family backgrounds and economic base. This vividly reflects in the scene of Elizabeth Bennet refusing Mr. Collins’ proposal. When Mr. Collins proposes to Elizabeth, she answers,

    “I thank you again and again for the honor you have done me in your proposals, but to accept them is absolutely impossible. My feelings in every 1respect forbid it.”

     These words show Elizabeth the determination that she prefers to keep single in all her life rather than to marry to Mr. Collins without love, and therefore, establish a romantic character.

     Secondly, her transitional role also displays in her writing style. That is to say, on the one hand, she inherited some characteristics of the neoclassicism in style and painted the world she knew; so it is foolish to expect from her the high-flown sentiment; but on the other hand, to some degree, her style is also different from the previous enlightening writers, like Fielding, just as she herself claimed that she not only

    painted the world she lived in with fidelity, but also with sympathy; with a sensitive sense of its blemishes, but also with a true insight into its redeeming virtues. So her characters evolve themselves without any great dramatic episodes. And although her language sparkles with wit and irony, they are moderate. Her novel Pride and Prejudice provides a good example for this point. We know that Mr. Collins is a clergyman in this novel. When he sings high praise for his patroness, he says,

     “Lady Catherine was reckoned proud by many people, he knew, but he had never seen anything but affability in her. She had always spoken to him as she would to any other gentleman; she made not the smallest objection to his joining in the society of the neighborhood, nor to his leaving his parish occasionally for 2a week or two to visit his relations.”

    It is true that these words are also the satire to Mr. Collins’ snobbery. But different from the traditional satire, Jane Austen is too conscious of people’s snobbery to be angry with them, and thus gives her criticism a flavor of humor. Therefore, we say that in style she is also a transitional writer at the turn of the century, just as one of the most important neoclassicist, Sir Walter Scott, comments on her,

     “The big bow-wow strain I can do myself, like any now going; but the exquisite touch which renders ordinary commonplace things and characters interesting from the truth of the description and the sentiment is denied 3me.”

     To sum up, through the above analysis, we can make such a summary that Jane Austen plays a transitional role in the English literature from the 18th neoclassicism to the 19th romanticism. Trained in the enlightening tradition, she displays the feeling moderately; influenced by the coming romanticism, she satirizes the society in comedy. So it might be an error to say that Jane Austen is incapable of sentiment, because nobady can create a true comedy of life without romance and passion that is even in the humblest existence.

    Synthesis and characterizations of amorphous titania


     Abstract: Using tetrabutyl titanate as a precursor, amorphous titania (TiO2) nanoparticles have beensynthesized by a sol-gel method. Thermal gravimetry, differential scanning calorimetry, powder X-raydiffraction, fourier transformed infrared spectroscopy, transmission electron microscopy, and X-rayphotoelectron spectroscopy were employed to characterize the amorphous titania nanoparticles. Theresults indicated that after calcination at 320?C for 2 hours, the product obtained is pure,

    amorphousTiO2 nanoparticle aggregates of about 30 nm in diameter with the primary particle size of 3-5 nm.

     Key words: Nanoparticles; Titania; Amorphous particels

     0 Introduction

     Titania (TiO2) is an important metal-oxide semiconductor and has been extensively studied ascatalyst supports,[1] semiconductors,[2] materials for solar-energy conversion,[3, 4] photocatalysis,[5,6] gas sensors,[7] and electrochemical capacitors.[8] For the synthesis of crystalline titania particles,a number of chemical methods including the sol-gel method,[9] hydrothermal synthesis,[10]reversed micelle method,[11] cathodic electrodeposition,[12] and chemical vapor deposition[13] wereemployed. Usually, conventional titania precursors such as titanium (IV) chloride (TiCl4) andtitanium alkyloxide [Ti(OR)4] were used for preparation of TiO2.[14]

     Because of the lack of long range order and the metastability in thermodynamics, amorphousTiO2 materials will exhibit novel properties. For example, amorphous TiO2 films showed a highdielectric constant and might be candidates as the dielectric layer in ultra-thin film capacitors.[15]Amorphous TiO2 films exhibited super-hydrophilicity which is crucial in self-cleaningtechniques.[16, 17] Amorphous TiO2 has shown potential applications as electrodes in solarbatteries[18] and in semiconductors.[19] Computational modeling of amorphous TiO2 showed thatthe structural characteristics of amorphous TiO2 nanoparticles were different from that of theamorphous TiO2 bulk.[20, 21] Zhang et al. synthesized amorphous titania by hydrolysis of titaniumethoxide at the ice point; the synthesized titania consisted of 0.2 μm TiO2

    aggregates.[22] However,amorphous TiO2 nanoparticles with aggregate sizes in nanoscale have not been reported so far.

    In our present letter, we synthesized amorphous TiO2 nanoparticles using tetrabutyl titanate asthe precursor by a sol-gel method and subsequent calcination. The products obtained werecharacterized by thermal

    gravimetry (TG), differential scanning calorimetry (DSC), powder X-raydiffraction (XRD), Fourier transformed infrared spectroscopy (FTIR), transmission electronmicroscopy (TEM), and X-ray photoelectron spectroscopy (XPS).

     1 Experiments

     Amorphous titania nanoparticles were prepared from tetrabutyl titanate [Ti(OC4H9)4, TBOT],ethanol, and water by a sol-gel method. The overall reaction for the conversion of tetrabutyltitanate to titania isReaction (1) can be divided into two separate reactions,[23, 24] namely,

     In the present study, R is C4H9. Since an exchange reaction can occur between the ethanolsolvent and the tetrabutyl-based reactant, the final product can be an oxo-alkoxide as shown inreaction (3) if the hydrolysis reaction is limited, or can be titania if the hydrolysis reaction iscomplete (x=4) and all the organic (R) groups are removed from the starting alkoxide. Thesereactions, as well as other theoretical and experimental results of the hydrolysis of titaniumalkoxides, have been reviewed by Bradley et al.[25]

     Firstly, the solution S1 [a mixture of 5 ml Ti(OC4H9)4 and 45 ml C2H5OH (Eth)] and thesolution S2 (a mixture of 50 ml C2H5OH and 50 ml H2O) were prepared. Then, with the solutionS2 being vigorous stirred by an electric magnetic stirrer, the solution S1 was slowly dripped intothe solution S2 at 2 drops per second. After the dripping process was complete, the solution wasmixed by the electric magnetic stirrer for 12 h; and the titania particles precipitated in the solution.Then the resulting precipitates were vacuum filtered. The resulting precipitates were washed withethanol by resuspending the precipitate in ethanol and filtering. The washed precipitates weredried at 100?C for 6 h and calcined at 320?C for 2 h.

    Thermal analysis (TG and DSC) was performed on the as-synthesized titania precursorsample using a Netzsch STA 449C TA instrument in flowing nitrogen atmosphere at a heating rateof 10?C/min. XRD analysis was performed on Rigaku D/Max-2400 X-ray diffractometer with CuKα radiation to determine

    the structure of the samples. To provide direct evidence of TiO2, FTIRspectra were recorded in the range of 400~4000 cm-1 at room temperature (RT) by using a Nexus670 spectrometer. The samples for FTIR were prepared using the KBr technique which werecalibrated by polystyrene. XPS measurements were carried out on a PHI-5702 (PhysicalElectronics, Inc.) spectrometer. During the XPS analysis, Al Kα X-ray beam with a power

    of 250W was adopted. The vacuum of the instrument chamber was 1?10-7 Pa.

    The binding energy wascalibrated with reference to C 1s peak (285.0 eV). TEM observations were conducted on a JEM2010 electron microscope operated at 200 kV. Energy dispersive X-ray spectroscopy (EDS)analysis was conducted on the TEM during the TEM observations The TEM samples wereprepared by depositing the powder suspended into ethanol onto a holey

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