• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    小鼠神經(jīng)再生的檢測(下)

    2013-11-27 08:23:32潘寶晗約翰格里芬邁克波里保羅霍夫曼默汗默德H法拉
    關(guān)鍵詞:約翰霍普金斯巴爾的摩馬里蘭州

    潘寶晗,約翰 W.格里芬,2,4,邁克 A.波里,保羅 N.霍夫曼,3,默汗默德H.法拉

    (1.約翰霍普金斯醫(yī)學(xué)院 神經(jīng)科,美國 馬里蘭州巴爾的摩21205;2.約翰霍普金斯醫(yī)學(xué)院 神經(jīng)科學(xué)系,美國 馬里蘭州巴爾的摩21205;3.約翰霍普金斯醫(yī)學(xué)院 眼科,美國 馬里蘭州巴爾的摩21205;4.約翰霍普金斯醫(yī)學(xué)院 病理科,美國 馬里蘭州巴爾的摩21205;)

    4 Histological measures of nerve regeneration

    In the mouse sciatic system faster growing sprouts outgrow the available length of nerve within 1week,so that data collection is restricted to that interval,must be done in daily increments,and requires assessment of short nerve segments of 1mm or less.For these reasons histological approaches that identify the regenerating fibers in longitudinal sections of the whole nerve or in whole mounts of the nerve are most satisfactory.Immunostaining for axonal markers can be useful in measuring the position of the fast growing axons at early times after injury in the mouse.With some antibodies penetration of the whole nerve is adequate,so that immunostaining of whole mounts of nerves can be done and no section-to-section variation is introduced.Optical sections of whole mounts can be made by confocal microscopy.Alternatively,the nerves can be examined in longitudinal sections.

    The best markers are the products of genes that are rapidly and markedly upregulated by axotomy.The growth-associated protein GAP43is the most widely used of these.GAP43is a highly regulated cytoplasmic protein that is normally expressed at low levels and in only some peripheral axons,including the nociceptors innervating the epidermis.After axotomy,it is induced in the nerve cell body[29],rapidly transported down the axon,(Fig.7)and inserted into and retained within lipid rafts in the growth cone by dual palmitoylation(Nakamura et al.,1998).It is a PKC substrate.Its phosphorylated form interacts with a number of terminal components,and acts to cap actin filaments.For our present purposes the fact that it is largely absent from the axon before injury,that it becomes abundant rapidly after axotomy,that it is rapidly transported so that it accumulates within the growth cones even in the longest nerves of mice within short periods after synthesis,and that there are excellent antibodies to detect it immunohistochemi cally combine to make it a superb marker for studies of regeneration(Fig.7).

    Fig.7 Immunostaining for GAP43 3 days after sciatic nerve crush.The axons in the proximal stump are intensely stained for the marker,which identifies sprouts that are beginning distal to the crush site.

    Endogenous axonal proteins that are normally present in the axon,including neurofllament proteins and class IIIβ-tubulin,can be used to identify axonal sprouts.However,they have an inherent limitation in small animals because the axons contain these markers before injury.These markers identify axonal debris during the early stages of Wallerian degeneration,making the distinction from regenerating sprouts in the distal stump tenuous.This concern also applies to fluorescent proteins(XFP variants of the green fluorescent protein)con-trolled by neuronal promoters such as Thy1.2.Fig.5Aillustrates the appearance of the sciatic nerve 5 days after sciatic nerve transection in a line H YFP mouse.Because the Schwann cells of the denervated Bunger bands contain breakdown products of the axoplasm,they are fluorescent,and in this setting it is difficult to discriminate the regenerating axons from the residua of interrupted axons(Fig.5B).

    4.1 Low molecular weight fluorescent markers introduced at sites of axotomy

    Measurement of the lengths of regenerating sprouts in longitu-dinal sections of nerve can also be done following the injection of axonally transported compounds into the nerve proximal to the site of nerve injury.The strategy in these methods is to crush the proximal stump of a regenerating nerve above the site of the previous axotomy and then to inject the marker into the endoneurial space.Taken up into axons of the newly created distal stump,the marker passes the original site of axotomy and then to fills out the regenerated fibers(Fig.8).The biotin derivative biotinamide hydrochloride,or Neurobiotin(NB)has been shown to be a useful marker for intracellular tracing in slice preparations after electro-physiological recording of single living neurons[30].This compound has a very low molecular weight of 323Da and is transported in both retrograde and anterograde directions depending on the method of injection and the type of tissue.The rate of transport of this tracer is high[30].In addition,this compound has no signs of toxicity in any labeled neurons and significant effects on neuron membrane properties have not been identified.In mouse sciatic nerves the available length of nerve below a crush can be filled out within b 4hafter injection.This method is advantageous for identifying the transition from latency to outgrowth and for the early stages of outgrowth,because it does not depend on the induction of new neuronal proteins,as required for GAP43,and because for the most part it does not identify marker in axons undergoing Wallerian degeneration,just discussed.How ever,we have found that,when injected 48hafter the initial sciatic nerve crush,4hlater a percentage of fibers undergoing Wallerian degeneration in the distal stump is labeled.This is likely to represent uptake into axons that were still in continuity at the time of injection,but then entered the phase of explosive degradation of axoplasm and axonal segmentation within the next 4h.For this reason interpretation of data from early studies done the first few days after the nerve injury must be done cautiously.

    Fig.8 Longitudinal section of a neurobiotin-injected mouse sciatic nerve 5 days after nerve crush.The arrow identifies the crush site.Neurobiotin was injected at the site of a more proximal nerve crush 4 h before the nerve was removed.The sprouts in the distal stump are well displayed.

    An alternative to these measures,all of which are based on longitudinal sections of the distal stump,are assessments of the numbers of fibers reaching a defined point distal to the crush at a specific time,as determined in transverse sections of plastic-embedded specimens.Because at the light level the number of myelinated fibers can be reliably counted,this measure appears foolproof,but there are several cautions in the interpretation of this data.Electron microscopy is required to assess premyelinated and unmyelinated sprouts.Importantly,myelinated parent axons can extend many sprouts that myelinate before reaching their targets.Thus counting only myelinated fibers may test the extent of axonal branching or the speed of myelination rather than the extent of reinnervation of targets.The accuracy of these axonal counts as a measure of regeneration also depends on having the specimens embedded in known proximal-distal orientation.I-magine that the front of regenerating sprouts on day 4after nerve crush has reached 6.5mm from the crush site.If the fixed mouse sciatic nerve is divided into seven 2-mm blocks,the site of interest will be the proximal face of the fourth block distal to the crush.If this nerve segment is cut on its distal face few sprouts will be seen.If in a second experimental condition with no biological difference in regeneration the block face was oriented with the proximal face forward and sprouts were seen the calculation might be made that the condition produced a 25%increase in the rate of regeneration.Such errors should wash out with large numbers of animals,but the data would remain unnecessarily noisy.An alternative to protect against this error is to cut every block to the center before taking sections for analysis.

    4.2 Retrograde tracing

    Various kinds of tracers have been developed as the fundamental methods to map connectivity in the nervous system.Aspects to be considered in selection of a tracer for measuring regeneration include:anterograde or retrograde tracing methods,uptake mechanisms,transport speed,toxicity and stability.To obtain optimal results,it is important to choose the tracer best suited for the system studied and for the specific question asked.

    Retrograde tracing to label neurons regenerating into specific nerves or reaching specific targets is a well established approach that remains highly valuable.HRP has been used to analyze the consequences of peripheral nerve injury and to evaluate regeneration rates.Other tracers such as Fluoro-Gold and a series of fluorescent compounds,as well as biotinylated dextran amine [BDA],have been frequently used to retrogradely label motor neurons in the spinal cord.The nerves can be transected at a distal site and the cut end“dipped”in the retrograde marker,or with some of the markers they can be injected into the target(for example,a specific muscle or a foot pad).These techniques require practice and controls for diffusion and other technical concerns,but they can be used effectively and highly reproducible.Concerns include determining the degree of spread from the injection site and the fact that some of the retrograde markers produce longterm toxicity in the neurons that contain them.

    5 Electrophysiological measures

    In larger animals,it is possible to measure the progress of sprouts down the nerve by near-needle techniques.Such an approach can be adapted to the mouse in terminal in vitro or in in vivo studies.For in vitro analysis,the nerve is removed from above the site of the crush or repair to the most distal site available and hung on bipolar electrodes in an appropriate oxygenated salt solution.Both A and C waves can be detected and the amplitudes measured.The rate of recovery of A fiber and C fiber compound action potential(CAP)amplitudes,compared to u-ninjured control nerves,gives a useful measure of regeneration to the site of distal transection.In vivo electrophysiological measures can be used as time-to-target measures.

    5.1 Radiolabeled axonal markers

    Now rarely used,radioisotopic label of the fast anterograde component of axonal transport can give an efficient and vivid picture of the distribution of growth cones distal to a nerve injury.The technique depends upon the fact that anterogradely transported proteins accumulate in the cut ends of severed nerve fibers and,as the fibers regenerate,in the growth cones.Injection of labeled isotopes such as S35-methionine into the ventral horn or dorsal root ganglia labels fast axonal transport.In uninjured nerves the “peak”of pulse-labeled fast transport passed down the nerve to the nerve endings,and a relatively flat“shoulder”of radioactivity remains along the nerve.If the nerve is crushed a few days before labeling the radioactivity accumulates within the growth cones,so that when the distribution of radioactivity is plotted the relative position of the fastest growing fibers,and an estimate of the proportion growing at that rate can be determined[31].In young animals the front of radioactivity passes down the nerve as a sharply defined peak,reflecting the fact that many nerve fibers grow at rates approaching that of the fastest fibers.In older animals the proportion growing at slower rates increases,and more radioactivity remains near the crush site,reflecting fibers that have grown very little.The limitation to this approach is the degree of nonspe-cific radioactive contamination of nerves that results from the isotope injection.In the rat this is negligible,but in the mouse non-transported radioactivity can be a limiting confound.

    5.2 Measurement of collateral sprouting and reinnervation

    Collateral sprouting leading to reinnervation of denervated targets has arguably received insufficient research attention in relation to its potential therapeutic importance.In patients with proximal nerve injuries a realistic goal is to restore protective sensibility to a partially denervated foot or hand by collateral spouting of uninjured fibers.In the rodent collateral sprouting of nociceptors can result in recovery of protective sensibility in denervated skin[32].As with regeneration of interrupted fibers,rodents are much better at generating collateral sprouts than humans[33].However,the mechanisms underlying collateral sprouting may be more amenable to therapeutic manipulation in man than are those required for regeneration of interrupted fibers.The magnitude of scientifi advance required to improve collateral sprouting is likely to be less than that required for long distance regeneration of the interrupted fibers.

    As noted,throughout this discussion collateral sprouting is used in the restricted definition to describe the process by which intact,uninjured nerve fibers extend axonal branches into the regions previously occupied by other nerve fibers that have undergone degeneration.What fiber types undergo collateral growth Unmyelinated fibers are well recognized for this capacity.Aβmechanosensory fibers can sprout.For Aβmyelinated fibers the evidence is mixed.Without question,sprouting to innervate nearby denervated muscles can occur from the nodes of uninjured myelinated motor fibers above the motor nerve terminals.Convincing physiological evidence also indicates that Aβmechan-osensitive afferents that innervate teeth can sprout in the jaw,and can even cross the midline to innervate teeth from the other hemijaw.However,this is decidedly the exception.The reason for the paucity of collateral growth from myelinated fibers may reflect the growth inhibitory properties of many of the consti-tuents of myelin-forming Schwann cells.In the myelin sheath,in the attachment sites of the paranode,and in the periaxonal membrane of the internode are at least two proteins with growth inhibitory potential,MAG and netrin-1.In addition,there is immunocytochemical data showing that OMgP is found in the Schwann cell fingers overlying the nodes[34].These molecules are likely to restrict collateral sprouting in fibers with intact nodes.

    Extensive studies of collateral sprouting of uninjured C and Aβsensory fibers,as well as unmyelinated postganglionic sympathetic fibers,have demonstrated reinnervation of neighboring denervated skin.This has been satisfyingly detailed in the advantageous system of the cutaneous nerves of the thorax[35].The dorsal and lateral cutaneous nerves provide sensory innervation of the back.Section of one of these cutaneous nerves leaves an island without nociception or mechanoception.With collateral sprouting from neighboring segments sensation can restored.Conversely,section of several of these cutaneous nerves with sparing of only one results in an island of preserved sensibility that gradually expands as a result of because of collateral sprouting.The size of this island can be mapped by pinch or heat pain stimulation of the back skin to elicit the cutaneus trunci reflex.In this preparation,nociceptors undergo collateral sprouting that has been documented by electron microscopy.Strikingly,no Aβ fibers extended beyond the baseline boundaries of the island.

    Comparable results have been achieved in the rodent foot.For example,following sciatic nerve ligation,the saphenous nerve sprouts into the sciatic distribution(Fig.6D).Again,Aβfibers are minor participants.However,in some other model systems Aβsprouting has been adduced by electrophysiological evidence.For example,collateral sprouting of trigeminal fibers across the midline of the jaw to innervate the dental pulp has been shown for Aβas well as nociceptor fibers.Some of these fibers can cross the midline of the jaw to reach denervated teeth.In the rat hindfoot,Kinman and Aldskogius[36]found that regeneration of the saphenous into the territory of the sciatic nerve was speeded by prior crush of the sciatic,suggesting that a“growth state”in the collaterally growing nerve cell speeded outgrowth.Bajrovic and Sketelj reached the opposite conclusion in studies in which the peroneal nerve was spared and the saphenous,sural,and tibial were cut.In this study either simultaneous or earlier crush of the peroneal nerve slowed regrowth into the denervated territories.The reasons for this striking discrepancy are unclear.

    The Benowitz lab has shown that a focal injection of inosine,a metabolite of adenosine that activates N-kinase,is sufficient to inducecollateral sprouting from uninjured CNS fibers.The best defined drivers of collateral sprouting are growth factors.In models in which anti-NGF strategies have been used collateral sprouting is eliminated.In vitro studies using NGF-coated beads have formally shown that a gradient of a relevant growth factor along the shaft of an axon is sufficient to induce accumulation of actin and mitochondria[37]at the point nearest the bead,followed by extension of a sprout toward the bead.In vivo,a focal injection of cells expressing NGF under lentiviral control is suf?cient to induce collateral sprouting of medial septal fibers.

    Denervated Schwann cells in the subepidermal plexus are a source of growth factors,and at least contribute to the measured increases in growth factors in denervated skin.It is likely that the basal epidermis is also a site of elevated growth factor production.Consistent with this is the observation of the responses of epidermal fibers to denervation of neighboring regions.A simple system is excision of a cylinder of skin(dermis and epidermis)by apunch of the type used in diagnostic biopsies[38].The site is rapidly covered by new epidermis.The dermal defect is filled by a connective tissue plug.The epidermal fibers on the margin of the incision begin within days to elongate at their endings(ultraterminal sprouting),and incline toward the new epidermis.They may in part be carried by the migration of keratinocytes into the defect,but the inclination persists for weeks.In addition some of these“tilted”fibers extend collaterals that grow into the new epidermis.These sprouts typically drop to the dermalepidermal junction and grow just on the epidermal side of the basal epider-mis.In man their growth is slow,but within months they innervate the entirety of a 3mm defect.These sprouts in turn send collaterals vertically into the epidermis,and this arrangement is maintained for months.

    An “incision”model provides an instructive contrast.In this model,the skin punch is passed into the skin to produce a circular incision,but no skin is excised.The ultraterminal sprouting and growth along the dermalepidermal basal lamina occurs as in the excision model.The important difference in the preparation is that a dermal column topped by the original epidermis survives.Because the incision transected the subepidermal network of nerve fibers,both the epidermis and the dermis with its Schwann cell bands survive.Regenerating axons,at least in part from the transected ends of the fibers in the subepidermal network,grow into the dermis and grow up to the epidermis.As these regenerating fibers enter epidermal regions occupied by the collateral and ultraterminal sprouts they quickly out-compete them,so that the extended a-xons return to the borders of the incision[39].This observation suggests that epidermal axons can only maintain expanded terminal arbors if they have uncompeted access to growth factors from the fullfield.This in turn suggests that the relatively regular spacing of fibers in the normal epidermis may be determined by the available growth factor resources.

    One of the most striking but relatively little explored enhancers of collateral sprouting appears to be electrical activity in the nerve.In the dorsal cutaneous nerve “islands”described above collateral reinnervation was accelerated by repeated mechanical stimulation of the skin within the innervated regions,as well as by electrical stimulation of the uninjured nerves.Electrical stimulation can also improve specificity of reinnervation by the sprouts of interrupted fibers.

    6 Conclusions

    Studies of peripheral nerve regeneration to this point have been dominated by measurements made in the sciatic system of the rat.Molecular genetic approaches will increasingly mandate the use of the mouse,and this transition will require in turn in-vestments of effort into developing improved measures of regeneration.These measures will complement anticipated advances in understanding the fundamental changes associated with the entry of the neuron into the axonal growth state,with the local mechanisms involved in axonal building during regeneration,the manipulation of growth factors and growth factor signaling,the roles of Schwann cells,and the clearance of myelin debris.The same functional,behavioral,electrophysiological,and histological tools are likely to be useful in the study of axonal degeneration and axonal protection in experimental neuropathies.

    New model systems of regeneration in specific nerves will complement the established models using sciatic nerve regeneration.For many needs the use of tools that specifically assess muscle reinnervation,the reinnervation of the various sensory transducers,and reinnervation of autonomic targets are likely to become feasible.The correlation of reinnervation to function will depend in part on reestablishing the specificity of the connections.Measurements that are adapted for the mouse and that can assess the latency to growth,the rate of axonal growth,the specificity of reinnervation,and the extent of functional recovery will be particularly valuable.

    Finally,it seems likely that increased attention will be paid to approaches that amplify collateral sprouting and reinnervation.Collateral sprouting can lead to functional improvement in strength of partially denervated muscle and to improvement in protective sensibility.Importantly,these functional improvements can occur without the need for long distance nerve fiber growth that regeneration of interrupted fibers may entail.Manipulation of growth factor signaling and possibly electrical stimulation of uninjured nerves have been shown in rodents to accelerate collateral growth,and are likely to be among the approaches that will be further tested for this purpose.

    Acknowledgements

    Funding for studies from the authors’laboratories was provided by the Adelson Program in Neural Repair and Rehabilitation of the Adelson Medical Research Foundation,the Robert Packard Center for ALS Research,the Muscular Dystrophy Association,and the NIH NINDS.Mr.Vamsi Kalari provided Figure 7.

    [29]Hahm k,Sirdofsky M,Browen A,et al.Collateral sprouting of human epidermal nerve fibers following intracutaneous axotomy[J].J Peripher Nerv Syst,2006,11(2):142-147.

    [30]Kita H,Armstrong W.A biotin-containing compound N-(2-aminoethyl)biotinamide for intracellular labeling and neuronal tracing studies:comparison with biocytin[J].J Neurosci Methods,1991,37(2):141-150.

    [31]Hoffman P N,Lasek R J.Axonal transport of the cytoskeleton in regenerating neurons:constancy and change[J].Brain Res,1980,202(2):317-333.

    [32]Jackson P C,Diamond J.Regenerating axons reclaim sensory targets from collateral nerve sprouts[J].Science,1981,214(4 523):926-928.

    [33]Healy C,LeQuesne P M,Lynn B.Collateral sprouting of cutaneous nerves in man[J].Brain,1996,119(966):2 063-2 072.

    [34]Kinman E,Aldskogius H.Collateral sprouting of sensory axons in the glabrous skin of the hindpaw after chronic sciatic nerve lesion in adult and neonatal rats:a morphological study[J].Brain Res,1986,(377):73-82.

    [35]Huang J K,Phillips G R,Roth A D,et al.Glial membranes at the node of Ranvier prevent neurite outgrowth[J].Science,2005,310(5 755):1 813-1 817.

    [36]Inserra M M,Bloch D A,Terris D J.Functional indices for sciatic,peroneal,and posterior tibial nerve lesions in the mouse [J].Microsurgery,1998,18(2):119-124.

    [37]Jacquin M F,Hu J W,Sessle B J,et al.Intra-axonal Neurobiotin injection rapidly stains the long-range projections of identi?ed trigeminal primary afferents in vivo:comparisons with HRP and PHA-L[J].J Neurosci Methods,1992,45(1-2):71-86.

    [38]Kong L,Wang X,Choe D W,et al.Impaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice[J].J Neurosci,2009,29(3):842-851.

    [39]Laird F M,F(xiàn)arah M H,Ackerley S,et al.Motor neuron disease occurring in a mutant dynactin mouse model is characterized by defects in vesicular trafficking[J].J Neurosci,2008,28(9):1 997-2 005.

    猜你喜歡
    約翰霍普金斯巴爾的摩馬里蘭州
    坍塌
    剃光頭,老三歲
    Bone Research近期被多種國際知名數(shù)據(jù)庫收錄
    環(huán)球時報(2014-03-31)2014-03-31 09:33:35
    做手邊清楚的事
    拖家?guī)Э? 去應(yīng)聘
    97人妻精品一区二区三区麻豆| 非洲黑人性xxxx精品又粗又长| 亚洲国产av新网站| 99久久九九国产精品国产免费| 在线 av 中文字幕| 97超视频在线观看视频| 人妻夜夜爽99麻豆av| 国产亚洲av片在线观看秒播厂 | 我要看日韩黄色一级片| 中文欧美无线码| 麻豆成人av视频| 亚洲国产成人一精品久久久| 成人无遮挡网站| 亚洲精品乱久久久久久| 1000部很黄的大片| 伦精品一区二区三区| 亚洲久久久久久中文字幕| 亚洲婷婷狠狠爱综合网| 777米奇影视久久| 国产精品国产三级专区第一集| 久久久久久久久中文| 久久精品夜色国产| 亚洲国产精品成人综合色| 免费电影在线观看免费观看| 麻豆久久精品国产亚洲av| 天堂av国产一区二区熟女人妻| 成人鲁丝片一二三区免费| 国产精品精品国产色婷婷| 国产精品av视频在线免费观看| 国内精品美女久久久久久| 国产又色又爽无遮挡免| 国产精品伦人一区二区| 亚洲国产最新在线播放| 少妇的逼好多水| 国产精品国产三级专区第一集| 国产男女超爽视频在线观看| av专区在线播放| 亚洲一区高清亚洲精品| 天天躁日日操中文字幕| 欧美精品国产亚洲| 国产亚洲5aaaaa淫片| 色综合色国产| 可以在线观看毛片的网站| 美女高潮的动态| 国产男人的电影天堂91| 少妇熟女aⅴ在线视频| 国产 一区 欧美 日韩| 精品人妻熟女av久视频| 舔av片在线| 免费播放大片免费观看视频在线观看| 国产黄片美女视频| 国产淫语在线视频| 丝瓜视频免费看黄片| 精品一区二区三区视频在线| 精品久久国产蜜桃| 3wmmmm亚洲av在线观看| 成年女人看的毛片在线观看| 欧美变态另类bdsm刘玥| 亚洲图色成人| 三级国产精品片| 能在线免费看毛片的网站| 精品久久久久久久久av| 久久久久精品久久久久真实原创| 边亲边吃奶的免费视频| 日本熟妇午夜| 成人欧美大片| 亚洲精品乱码久久久v下载方式| 在线免费十八禁| av卡一久久| 我要看日韩黄色一级片| 亚洲av男天堂| 亚洲av中文av极速乱| 高清午夜精品一区二区三区| 中国美白少妇内射xxxbb| 国产久久久一区二区三区| a级毛色黄片| 在线 av 中文字幕| 日日摸夜夜添夜夜爱| 精品久久国产蜜桃| 免费av毛片视频| av播播在线观看一区| 九九在线视频观看精品| 久久精品国产亚洲av涩爱| 成人美女网站在线观看视频| 成人二区视频| 好男人在线观看高清免费视频| 日韩av在线大香蕉| 夫妻午夜视频| 亚洲自偷自拍三级| 少妇被粗大猛烈的视频| 1000部很黄的大片| 欧美日韩综合久久久久久| 亚洲无线观看免费| 国产成人a区在线观看| 精品久久久久久成人av| 国产亚洲一区二区精品| 一级毛片我不卡| 两个人视频免费观看高清| 淫秽高清视频在线观看| 亚洲av.av天堂| 国产一区二区在线观看日韩| 午夜日本视频在线| 一级毛片黄色毛片免费观看视频| 天天一区二区日本电影三级| 午夜免费男女啪啪视频观看| 老师上课跳d突然被开到最大视频| 成年免费大片在线观看| 成人av在线播放网站| av卡一久久| 午夜亚洲福利在线播放| 日韩强制内射视频| 777米奇影视久久| 搡女人真爽免费视频火全软件| 蜜臀久久99精品久久宅男| 天堂中文最新版在线下载 | 国内精品宾馆在线| 能在线免费看毛片的网站| 一个人观看的视频www高清免费观看| 精品一区在线观看国产| 国产欧美另类精品又又久久亚洲欧美| 亚洲美女视频黄频| 国产精品福利在线免费观看| 成人毛片a级毛片在线播放| 欧美三级亚洲精品| 亚洲色图av天堂| 欧美激情国产日韩精品一区| 精品国内亚洲2022精品成人| 最近最新中文字幕大全电影3| 狠狠精品人妻久久久久久综合| 非洲黑人性xxxx精品又粗又长| 简卡轻食公司| 亚洲最大成人手机在线| 一个人观看的视频www高清免费观看| 国产黄片视频在线免费观看| 男人爽女人下面视频在线观看| 日本免费a在线| 精品一区在线观看国产| 国产亚洲午夜精品一区二区久久 | 美女主播在线视频| 亚洲乱码一区二区免费版| 黄片无遮挡物在线观看| 国产成年人精品一区二区| 一级毛片久久久久久久久女| 美女cb高潮喷水在线观看| 能在线免费看毛片的网站| 丰满少妇做爰视频| 色播亚洲综合网| 一个人免费在线观看电影| 欧美日韩视频高清一区二区三区二| 丰满乱子伦码专区| 日韩av免费高清视频| 亚洲av福利一区| 免费电影在线观看免费观看| 亚洲av.av天堂| 久久久久久久久大av| 日韩精品有码人妻一区| 欧美bdsm另类| 97人妻精品一区二区三区麻豆| 国产精品无大码| 亚洲精品日韩av片在线观看| 黄片无遮挡物在线观看| 精品人妻视频免费看| 美女大奶头视频| 91aial.com中文字幕在线观看| 亚洲精品,欧美精品| 人人妻人人澡欧美一区二区| 男女边摸边吃奶| 日韩精品青青久久久久久| 久久精品人妻少妇| freevideosex欧美| 汤姆久久久久久久影院中文字幕 | 99热网站在线观看| 亚洲av成人精品一二三区| 九九久久精品国产亚洲av麻豆| 亚洲av成人精品一二三区| 免费播放大片免费观看视频在线观看| 亚洲欧洲国产日韩| 精品一区二区免费观看| 亚洲综合色惰| 国内揄拍国产精品人妻在线| 国内揄拍国产精品人妻在线| 亚洲综合色惰| 日韩大片免费观看网站| 精品午夜福利在线看| 亚洲av成人av| 午夜激情久久久久久久| 国产精品日韩av在线免费观看| 一级a做视频免费观看| 亚洲最大成人av| 最近中文字幕高清免费大全6| 免费观看性生交大片5| 色播亚洲综合网| 97热精品久久久久久| 一本一本综合久久| 人妻少妇偷人精品九色| 国产黄色视频一区二区在线观看| 最近的中文字幕免费完整| 亚洲精品第二区| 成人二区视频| 亚洲人成网站在线播| 中文字幕人妻熟人妻熟丝袜美| 高清在线视频一区二区三区| 国产日韩欧美在线精品| a级一级毛片免费在线观看| 亚洲欧美成人精品一区二区| 日本熟妇午夜| 夫妻性生交免费视频一级片| 免费观看在线日韩| 我的老师免费观看完整版| 亚洲精品日韩在线中文字幕| 亚洲国产精品sss在线观看| av国产免费在线观看| 久久99热这里只有精品18| 亚洲一级一片aⅴ在线观看| 日本猛色少妇xxxxx猛交久久| 一级毛片电影观看| 国产高清有码在线观看视频| 亚洲高清免费不卡视频| 日本欧美国产在线视频| 国产精品美女特级片免费视频播放器| 亚洲成人一二三区av| 国产乱人偷精品视频| 国产探花在线观看一区二区| 亚洲国产日韩欧美精品在线观看| 国产成人freesex在线| 精品国产露脸久久av麻豆 | 亚洲欧洲国产日韩| 国产成年人精品一区二区| 久久久精品欧美日韩精品| 日韩av在线大香蕉| 亚洲av免费高清在线观看| 99热这里只有是精品在线观看| 久久久久久久国产电影| 国产探花在线观看一区二区| 久久久久久久久中文| 国产白丝娇喘喷水9色精品| 亚洲av成人精品一二三区| 亚洲内射少妇av| 超碰av人人做人人爽久久| 午夜老司机福利剧场| 亚洲精品日本国产第一区| 亚洲精品中文字幕在线视频 | 国产黄色小视频在线观看| 亚洲精品影视一区二区三区av| 99久国产av精品国产电影| 成年免费大片在线观看| 精品久久久久久久久久久久久| 赤兔流量卡办理| 少妇高潮的动态图| 国产免费一级a男人的天堂| 国产一区二区三区av在线| 日日撸夜夜添| 一级二级三级毛片免费看| 欧美xxxx黑人xx丫x性爽| 国产黄色视频一区二区在线观看| 亚洲人成网站在线播| 日韩欧美一区视频在线观看 | 精品国内亚洲2022精品成人| 伊人久久精品亚洲午夜| 亚洲电影在线观看av| 黄片无遮挡物在线观看| 最近最新中文字幕免费大全7| 亚洲最大成人av| 能在线免费看毛片的网站| 免费在线观看成人毛片| av黄色大香蕉| 国产成人午夜福利电影在线观看| 人体艺术视频欧美日本| 国产极品天堂在线| 日韩大片免费观看网站| 国产精品女同一区二区软件| 美女cb高潮喷水在线观看| 女人被狂操c到高潮| 国产成人精品一,二区| 人妻一区二区av| 身体一侧抽搐| 日韩伦理黄色片| 一级毛片电影观看| 性色avwww在线观看| 国产乱来视频区| 国产一区有黄有色的免费视频 | 国产免费福利视频在线观看| 日本色播在线视频| 亚洲欧洲日产国产| 成人无遮挡网站| 免费看av在线观看网站| 中文字幕久久专区| 伦理电影大哥的女人| 汤姆久久久久久久影院中文字幕 | 激情 狠狠 欧美| 午夜免费激情av| 国产v大片淫在线免费观看| 日韩电影二区| 大陆偷拍与自拍| 人人妻人人澡欧美一区二区| 亚洲内射少妇av| 日韩欧美精品免费久久| 亚洲国产成人一精品久久久| 80岁老熟妇乱子伦牲交| 国产有黄有色有爽视频| 欧美日韩精品成人综合77777| 国国产精品蜜臀av免费| 一区二区三区乱码不卡18| 国产v大片淫在线免费观看| 天堂av国产一区二区熟女人妻| 99视频精品全部免费 在线| 哪个播放器可以免费观看大片| 久久人人爽人人爽人人片va| 一区二区三区免费毛片| 女人被狂操c到高潮| 午夜免费观看性视频| 成年女人看的毛片在线观看| 日韩伦理黄色片| 国产精品女同一区二区软件| 水蜜桃什么品种好| 国产色爽女视频免费观看| 国内精品一区二区在线观看| freevideosex欧美| 国产成人福利小说| 2021少妇久久久久久久久久久| 欧美潮喷喷水| 午夜福利在线观看免费完整高清在| 日本午夜av视频| 91久久精品国产一区二区三区| 99久久精品国产国产毛片| 蜜桃久久精品国产亚洲av| 91狼人影院| 久久精品人妻少妇| 五月玫瑰六月丁香| 丰满少妇做爰视频| 狠狠精品人妻久久久久久综合| 中国美白少妇内射xxxbb| 久久久久久九九精品二区国产| 免费电影在线观看免费观看| 国产免费视频播放在线视频 | 国产精品一区二区性色av| 色综合亚洲欧美另类图片| 校园人妻丝袜中文字幕| 三级国产精品片| 小蜜桃在线观看免费完整版高清| 国产成人精品久久久久久| 免费av毛片视频| 91精品伊人久久大香线蕉| 国产精品久久久久久久久免| 日日摸夜夜添夜夜添av毛片| 男女边摸边吃奶| 九色成人免费人妻av| 欧美人与善性xxx| 亚洲精品日韩在线中文字幕| 日韩av免费高清视频| 亚洲四区av| videos熟女内射| 三级男女做爰猛烈吃奶摸视频| 国产不卡一卡二| 啦啦啦中文免费视频观看日本| 成年免费大片在线观看| 亚洲人成网站在线观看播放| 午夜福利在线观看免费完整高清在| 国产精品久久久久久精品电影| 国产免费视频播放在线视频 | 男人舔奶头视频| 久久久久久国产a免费观看| 亚洲精品日本国产第一区| 99热这里只有是精品50| 亚洲av中文av极速乱| 18禁裸乳无遮挡免费网站照片| 国产探花在线观看一区二区| 日本色播在线视频| 亚洲婷婷狠狠爱综合网| 极品少妇高潮喷水抽搐| 波野结衣二区三区在线| 精品久久久久久电影网| 亚洲av免费高清在线观看| 日本三级黄在线观看| 国产精品1区2区在线观看.| 日韩精品有码人妻一区| 综合色丁香网| 蜜桃亚洲精品一区二区三区| 日日摸夜夜添夜夜添av毛片| 22中文网久久字幕| 舔av片在线| 熟女人妻精品中文字幕| 亚洲av不卡在线观看| 亚洲成色77777| 亚洲成人中文字幕在线播放| 视频中文字幕在线观看| 超碰97精品在线观看| 久久久欧美国产精品| 人人妻人人澡欧美一区二区| 国产精品女同一区二区软件| 极品教师在线视频| 亚洲,欧美,日韩| 久久久久精品久久久久真实原创| 天天躁日日操中文字幕| av线在线观看网站| 亚洲经典国产精华液单| 七月丁香在线播放| 五月天丁香电影| 免费黄频网站在线观看国产| 国产午夜福利久久久久久| 国产精品人妻久久久影院| 国产亚洲av嫩草精品影院| 午夜免费观看性视频| 1000部很黄的大片| 中文精品一卡2卡3卡4更新| 精品人妻熟女av久视频| 啦啦啦韩国在线观看视频| 国产极品天堂在线| 美女xxoo啪啪120秒动态图| 国产中年淑女户外野战色| 少妇熟女aⅴ在线视频| 免费观看a级毛片全部| 亚洲aⅴ乱码一区二区在线播放| 亚洲av在线观看美女高潮| 久久久久久久久久久免费av| 777米奇影视久久| 最近2019中文字幕mv第一页| 少妇的逼水好多| 91av网一区二区| 在线观看免费高清a一片| 熟妇人妻不卡中文字幕| 国产精品一区二区性色av| 中文精品一卡2卡3卡4更新| av女优亚洲男人天堂| 免费av观看视频| av播播在线观看一区| 精品久久久精品久久久| 极品少妇高潮喷水抽搐| 欧美xxⅹ黑人| 18禁在线播放成人免费| 啦啦啦啦在线视频资源| 精品不卡国产一区二区三区| 高清日韩中文字幕在线| 中文乱码字字幕精品一区二区三区 | 观看免费一级毛片| 精品熟女少妇av免费看| 狂野欧美激情性xxxx在线观看| 久久精品久久久久久久性| 成人高潮视频无遮挡免费网站| 日韩欧美三级三区| 亚洲av免费高清在线观看| 亚洲欧美成人精品一区二区| 免费观看a级毛片全部| 色播亚洲综合网| 美女被艹到高潮喷水动态| 亚洲精品亚洲一区二区| 一级毛片aaaaaa免费看小| 国产永久视频网站| 国产亚洲最大av| 精品99又大又爽又粗少妇毛片| 全区人妻精品视频| 日韩av免费高清视频| 久久久久久久国产电影| 国产精品1区2区在线观看.| 久久久久久国产a免费观看| 天堂俺去俺来也www色官网 | 国产极品天堂在线| 国产av国产精品国产| 老师上课跳d突然被开到最大视频| freevideosex欧美| 天美传媒精品一区二区| 国产大屁股一区二区在线视频| 国内少妇人妻偷人精品xxx网站| 中国国产av一级| 丝袜喷水一区| 纵有疾风起免费观看全集完整版 | 国产美女午夜福利| 91狼人影院| xxx大片免费视频| 精品久久国产蜜桃| 中文字幕制服av| 欧美三级亚洲精品| 欧美日韩视频高清一区二区三区二| 高清毛片免费看| 国产精品一区二区性色av| 两个人视频免费观看高清| 亚洲在线自拍视频| 亚洲精品日韩在线中文字幕| 欧美成人a在线观看| 夜夜爽夜夜爽视频| 亚洲怡红院男人天堂| 2021少妇久久久久久久久久久| 内射极品少妇av片p| 我的女老师完整版在线观看| 成人性生交大片免费视频hd| 白带黄色成豆腐渣| 亚洲av成人av| 久久久久久久久久久丰满| 国内精品美女久久久久久| 成人午夜高清在线视频| 久久久久久久亚洲中文字幕| 2022亚洲国产成人精品| 春色校园在线视频观看| 国产成人午夜福利电影在线观看| 亚洲一级一片aⅴ在线观看| 欧美xxxx性猛交bbbb| 国产高清不卡午夜福利| 久久精品综合一区二区三区| 久久久久久久国产电影| 亚洲精品日韩在线中文字幕| 亚洲aⅴ乱码一区二区在线播放| 亚洲精华国产精华液的使用体验| 少妇丰满av| 久久精品国产亚洲网站| 欧美日韩综合久久久久久| 日韩强制内射视频| 又黄又爽又刺激的免费视频.| 国产精品国产三级专区第一集| 老司机影院成人| 日本熟妇午夜| 舔av片在线| 尤物成人国产欧美一区二区三区| 亚洲欧美精品专区久久| 大香蕉97超碰在线| 亚洲在久久综合| a级毛片免费高清观看在线播放| 成年av动漫网址| 欧美日韩在线观看h| 午夜免费激情av| 欧美最新免费一区二区三区| 国产精品三级大全| 亚洲精品乱码久久久v下载方式| 亚洲精品视频女| 最后的刺客免费高清国语| 日韩,欧美,国产一区二区三区| 亚洲熟妇中文字幕五十中出| 午夜久久久久精精品| 亚洲真实伦在线观看| 亚洲成人久久爱视频| 久久久久久久久久久免费av| 国产综合精华液| av专区在线播放| 一本—道久久a久久精品蜜桃钙片 精品乱码久久久久久99久播 | 亚洲自拍偷在线| 在线 av 中文字幕| 欧美区成人在线视频| 少妇猛男粗大的猛烈进出视频 | 国产一区有黄有色的免费视频 | 色综合站精品国产| 亚洲人成网站高清观看| 美女主播在线视频| 麻豆精品久久久久久蜜桃| 亚洲欧美精品自产自拍| 欧美成人一区二区免费高清观看| 日日摸夜夜添夜夜添av毛片| a级毛片免费高清观看在线播放| 国产在线一区二区三区精| 亚洲欧洲国产日韩| 韩国av在线不卡| 久久热精品热| 美女cb高潮喷水在线观看| 免费av不卡在线播放| 国产精品女同一区二区软件| 成人午夜精彩视频在线观看| 日韩大片免费观看网站| 熟妇人妻不卡中文字幕| av国产免费在线观看| 午夜激情福利司机影院| 国产免费一级a男人的天堂| 一区二区三区免费毛片| h日本视频在线播放| 欧美日韩视频高清一区二区三区二| 97超碰精品成人国产| 亚州av有码| 国产成人精品一,二区| 亚洲美女视频黄频| 色尼玛亚洲综合影院| 日韩中字成人| 国产精品久久久久久久久免| 亚洲av国产av综合av卡| 乱系列少妇在线播放| 午夜福利在线观看免费完整高清在| 男插女下体视频免费在线播放| 国产精品一区二区三区四区免费观看| 一区二区三区四区激情视频| 97超碰精品成人国产| 青春草亚洲视频在线观看| 可以在线观看毛片的网站| 精品久久国产蜜桃| 男女那种视频在线观看| 精品午夜福利在线看| 精品人妻一区二区三区麻豆| 丰满人妻一区二区三区视频av| 亚洲av不卡在线观看| 最新中文字幕久久久久| 欧美 日韩 精品 国产| av免费观看日本| 女人被狂操c到高潮| av在线观看视频网站免费| 在线免费观看不下载黄p国产| 天天躁夜夜躁狠狠久久av| 精品人妻熟女av久视频| 人妻夜夜爽99麻豆av| 午夜爱爱视频在线播放| 寂寞人妻少妇视频99o| av女优亚洲男人天堂| 天堂中文最新版在线下载 | 婷婷色麻豆天堂久久| 三级毛片av免费| 午夜福利高清视频| 水蜜桃什么品种好| 男的添女的下面高潮视频| 乱系列少妇在线播放| 边亲边吃奶的免费视频| 亚洲成人久久爱视频| 国产精品综合久久久久久久免费| 精品一区二区三区视频在线| 三级国产精品欧美在线观看| 日本一二三区视频观看| 成人毛片60女人毛片免费| 日本欧美国产在线视频| 久久久久久九九精品二区国产| 国产片特级美女逼逼视频| 一区二区三区高清视频在线| 国产精品美女特级片免费视频播放器|