Immunohistochemical localization of neurocalcin in human sensory neurons and mechanoreceptors

The localization of neurocalcin in the developing and adult human peripheral nervous system (dorsal root and sympathetic ganglia (DRG, SG), and enteric nervous system (ENS)) was investigated using immunohistochemistry. A subpopulation of large-sized neurons in DRG of 9 and 12 weeks old embryos showed immunoreactivity (IR), whereas the sympathetic ganglia or enteric neurons did not. In adults, neurocalcin IR was restricted to a subpopulation of large (13%) and intermediate (15%) sized neurons in DRG. The protein was also found in muscular (67%) and cutaneous (12%) nerve fibers, as well as in the axons supplying muscular (muscle spindles, Golgi's tendon organs, and perimysial Pacinian corpuscles) and cutaneous (Meissner's but not Pacinian corpuscles) mechanoreceptors, as well as motor end-plates. Present results demonstrate that neurocalcin in both developing and adult humans can be used as a specific marker for a subpopulation of sensory neurons coupled to proprioception and touch, and for axons of motoneurons forming motor end-plates.     

Sensory innervation of the human palmar aponeurosis in healthy individuals and patients with palmar fibromatosis

The human palmar aponeurosis is involved in hand proprioception, and it contains different sensory corpuscle morphotypes that serve this role. In palmar fibromatosis (classically referred to as Dupuytren''s disease), the palmar aponeurosis undergoes fibrous structural changes that, presumably, also affect the nervous system, causing altered perception. We analysed the various sensory nerve formation morphotypes in the palmar aponeuroses of healthy subjects and patients with palmar fibromatosis. To do this, we used immunohistochemistry for corpuscular constituents and the putative mechanoproteins PIEZO2 and acid‐sensing ion channel 2. Free nerve endings and Golgi‐Mazzoni, Ruffini, paciniform and Pacinian corpuscles were identified in both the healthy and the pathological conditions. The densities of the free nerve endings and Golgi‐Mazzoni corpuscles were slightly increased in the pathological tissues. Furthermore, the Pacinian corpuscles were enlarged and displayed an altered shape. Finally, there was also morphological and immunohistochemical evidence of occasional denervation of the Pacinian corpuscles, although no increase in their number was observed. Both PIEZO2 and acid‐sensing ion channel 2 were absent from the altered corpuscles. These results indicate that the human palmar aponeurosis is richly innervated, and the free nerve endings and sensory corpuscles within the palmar aponeurosis undergo quantitative and qualitative changes in patients with palmar fibromatosis, which may explain the sensory alterations occasionally reported for this pathology.     

A comparative analysis of the encapsulated end-organs of mammalian skeletal muscles and of their sensory nerve endings

The encapsulated sensory endings of mammalian skeletal muscles are all mechanoreceptors. At the most basic functional level they serve as length sensors (muscle spindle primary and secondary endings), tension sensors (tendon organs), and pressure or vibration sensors (lamellated corpuscles). At a higher functional level, the differing roles of individual muscles in, for example, postural adjustment and locomotion might be expected to be reflected in characteristic complements of the various end‐organs, their sensory endings and afferent nerve fibres. This has previously been demonstrated with regard to the number of muscle‐spindle capsules; however, information on the other types of end‐organ, as well as the complements of primary and secondary endings of the spindles themselves, is sporadic and inconclusive regarding their comparative provision in different muscles. Our general conclusion that muscle‐specific variability in the provision of encapsulated sensory endings does exist demonstrates the necessity for the acquisition of more data of this type if we are to understand the underlying adaptive relationships between motor control and the structure and function of skeletal muscle. The present quantitative and comparative analysis of encapsulated muscle afferents is based on teased, silver‐impregnated preparations. We begin with a statistical analysis of the number and distribution of muscle‐spindle afferents in hind‐limb muscles of the cat, particularly tenuissimus. We show that: (i) taking account of the necessity for at least one primary ending to be present, muscles differ significantly in the mean number of additional afferents per spindle capsule; (ii) the frequency of occurrence of spindles with different sensory complements is consistent with a stochastic, rather than deterministic, developmental process; and (iii) notwithstanding the previous finding, there is a differential distribution of spindles intramuscularly such that the more complex ones tend to be located closer to the main divisions of the nerve. Next, based on a sample of tendon organs from several hind‐foot muscles of the cat, we demonstrate the existence in at least a large proportion of tendon organs of a structural substrate to account for multiple spike‐initiation sites and pacemaker switching, namely the distribution of sensory terminals supplied by the different first‐order branches of the Ib afferent to separate, parallel, tendinous compartments of individual tendon organs. We then show that the numbers of spindles, tendon organs and paciniform corpuscles vary independently in a sample of (mainly) hind‐foot muscles of the cat. Grouping muscles by anatomical region in the cat indicated the existence of a gradual proximo‐distal decline in the overall average size of the afferent complement of muscle spindles from axial through hind limb to intrinsic foot muscles, but with considerable muscle‐specific variability. Finally, we present some comparative data on muscle‐spindle afferent complements of rat, rabbit and guinea pig, one particularly notable feature being the high incidence of multiple primary endings in the rat.     

Movement-related skin strain associated with goal-oriented lip actions

 It is generally accepted that sensory input contributes to the generation of natural movements. In most motor systems, muscle spindles, tendon organs, joint receptors, and cutaneous mechanoreceptors may provide proprioceptive information. However, the perioral area of the human face lacks muscle spindles, tendon organs, and joint receptors and is therefore a model system for the study of cutaneous afferent contributions to proprioception. This investigation examined a series of skin strains associated with lower-lip movements in human subjects to determine if such strains, which serve as stimuli for cutaneous mechanoreceptors, may underlie proprioception in the face. The results suggested that strains associated with lower-lip movements were of sufficient magnitude to elicit cutaneous mechanoreceptor discharge, as shown in recent human microneurographic studies. Further, the magnitude of multiple strains was predictive of lower-lip movement endpoints. These results highlight the potential importance of cutaneous mechanoreceptors as putative proprioceptors. Received: 16 February 1998 / Accepted: 26 May 1998     

Development of mechanoreceptor numbers in embryonic chick-quail chimeras

Our experiments addressed the problem of the regulation of the number of mechanoreceptors by sensory axons and/or their peripheral target tissues. According to a previous study (Zelená et al. 1997) white leghorn chickens have more muscle spindles in the plantaris muscle (45.4+/-7.8; mean+/-SD) than the Japanese quail (35.3+/-4.8) and significantly more Herbst corpuscles in the crural region (380.0+/-85.0) than the quail (124.9+/-32.8). Embryonic chick-quail chimeras were therefore used as a model with distinct recombinations of the nerve supply and peripheral tissue for studying the developmental control of these mechanoreceptors. The chick host leg bud was replaced with a quail leg bud of equal age and vice versa on embryonic day 3, prior to the onset of innervation of the periphery. Shortly before hatching the chimeras were sacrificed and muscle spindles and Herbst corpuscles counted. Recombinations of chicken nerves with quail limb buds have shown that the richer nerve supply by chick Ia axons induced a significant increase in the number of muscle spindles in the plantaris muscles (55.5+/-13.4) of the grafted quail limb. In some instances, a similar increase in spindle numbers was also found in control legs grafted onto hosts of the same species. In the reverse type of chimera where chick embryo legs were grafted onto quail hosts, spindles developed in lower numbers (27.3+/-3.2). In that case the lower number of Ia axons in quail nerves induced a lower number of spindles in the chicken muscle. The numbers of Herbst corpuscles were, however, low in both types of chimera. Quail legs grafted onto host chick embryos contained 126.8+/-26.4 corpuscles, presumably due to a restrictive influence of the smaller crural area in the quail. Chick legs grafted onto quail hosts had only 99.6+/-34.1 crural corpuscles; the target area in chick embryo legs failed to attract more quail axons and/or to induce axonal sprouting. The developmental regulation of the number of the two types of mechanoreceptors examined in our study thus differ. While sensory axons appear to play the dominant role in the development of muscle spindles, their role seems to be restricted by hitherto unknown peripheral factors during the development of Herbst corpuscles.     

Distribution of sensory receptors in joints of the upper cervical column in the laboratory marsupial monodelphis domestica.

In order to investigate the sensory innervation, the upper cervical spine of a small laboratory marsupial (monodelphis domestica) was examined with serial section light microscopy and re-embedding of selected sections for electron microscopy. Large numbers of free nerve endings supplied by A delta- and C-fibres were found in the longitudinal ligaments and facet joint capsules. Electron microscopically, areas of direct contact between axon and collagen fibres of the surrounding connective tissue separated only by the basal lamina were observed. Such structural adaptations suggest mechanoreceptive or polymodal nociceptive functions. In addition, about 100 small lamellated corpuscles were found in the longitudinal ligaments mainly concentrated around the first intervertebral disk. Electron microscopy shows finger-like processes extending from the axon terminal into the inner core lamellae. These are the likely sites of the mechanoelectric transduction process. Smaller numbers of lamellated corpuscles were seen in the lower intervertebral disks and facet joint capsules. Lamellated corpuscles are known to function as rapidly adapting mechanoreceptors supplementing information supplied by muscle spindles to the CNS about position and movement of the cervical spine.     

Merkel cells and Meissner's corpuscles in human digital skin display Piezo2 immunoreactivity

The transformation of mechanical energy into electrical signals is the first step in mechanotransduction in the peripheral sensory nervous system and relies on the presence of mechanically gated ion channels within specialized sensory organs called mechanoreceptors. Piezo2 is a vertebrate stretch‐gated ion channel necessary for mechanosensitive channels in mammalian cells. Functionally, it is related to light touch, which has been detected in murine cutaneous Merkel cell–neurite complexes, Meissner‐like corpuscles and lanceolate nerve endings. To the best of our knowledge, the occurrence of Piezo2 in human cutaneous mechanoreceptors has never been investigated. Here, we used simple and double immunohistochemistry to investigate the occurrence of Piezo2 in human digital glabrous skin. Piezo2 immunoreactivity was detected in approximately 80% of morphologically and immunohistochemically characterized (cytokeratin 20⁺, chromogranin A⁺ and synaptophisin⁺) Merkel cells. Most of them were in close contact with Piezo2⁻ nerve fibre profiles. Moreover, the axon, but not the lamellar cells, of Meissner's corpuscles was also Piezo2⁺, but other mechanoreceptors, i.e. Pacinian or Ruffini's corpuscles, were devoid of immunoreactivity. Piezo2 was also observed in non‐nervous tissue, especially the basal keratinocytes, endothelial cells and sweat glands. The present results demonstrate the occurrence of Piezo2 in cutaneous sensory nerve formations that functionally work as slowly adapting (Merkel cells) and rapidly adapting (Meissner's corpuscles) low‐threshold mechanoreceptors and are related to fine and discriminative touch but not to vibration or hard touch. These data offer additional insight into the molecular basis of mechanosensing in humans.     

Development of Meissner‐like and Pacinian sensory corpuscles in the mouse demonstrated with specific markers for corpuscular constituents

The development of Meissner‐like and Pacinian corpuscles was studied in mice [from postnatal day (Pd) 0 to 42] by using immunohistochemistry for specific corpuscular constituents. The battery of antigens investigated included PGP 9.5 protein and neurofilaments, as markers for the central axon; S100 protein, vimentin, and p75^LNGFR protein, to show Schwann‐related cells; and epithelial membrane antigen to identify perineurial‐related cells. In Meissner‐like corpuscles immunoreactivity (IR) for neuronal markers was found by Pd7 and later. The lamellar cells of these corpuscles expressed first S100 protein IR (Pd7 to Pd42), then vimentin IR (Pd12 to Pd42), and transitory p75^LNGFR IR (Pd7 to Pd19–20). Vimentin IR, but not epithelial membrane antigen, was detected in the capsule‐like cells of the Meissner‐like corpuscles. On the other hand, the density of Meissner‐like corpuscles progressively increased from Pd0 to Pd19–20. Pacinian corpuscles were identified by Pd7. From this time to Pd42 the central axon showed IR for neuronal markers, and the inner core cells were immunoreactive for S100 protein. Moreover, vimentin IR was detected in the inner core cells by Pd19 and later. Unexpectedly, the central axons displayed S100 protein IR (from Pd7 to P28), while p75^LNGFR protein IR or epithelial membrane antigen IR were never detected. Taken together, and based on the expression of the assessed antigens alone, the present results suggest that the Meissner‐like and the Pacinian corpuscles in mice become mature around Pd19–Pd28 and Pd20, respectively. Furthermore, these results provide a baseline timetable for future studies in the normal or altered development of sensory corpuscles in mice since specific sensory corpuscles are functionally associated with different subtypes of sensory neurons the development of which is selectively disturbed in genetically manipulated mice. Anat Rec 258:235–242, 2000. © 2000 Wiley‐Liss, Inc.     

Localization of PIP5Kγ selectively in proprioceptive peripheral fields and also in sensory ganglionic satellite cells as well as neuronal cell membranes and their central terminals

Based on a previous study by others reporting that PIP5Kγ (phosphatidylinositol 4-phosphate 5-kinase γ) and its product, phosphatidylinositol 4,5 bisphosphate (PIP₂), are involved in the regulation of nociception, the present immunohistochemical study examined the localization of PIP5Kγ-immunoreactivity in dorsal root ganglia (DRG) and their peripheral and central terminal fields. PIP5Kγ-immunoreactivity was localized for the first time in the muscle spindles, in which it was found in I-bands of polar regions of intrafusal muscle fibers and also in sensory nerve terminals abutting on equatorial regions of the muscle fibers. This finding indicates the involvement of PIP5Kγ in the proprioception and suggests somehow complicated mechanisms of its involvement because of its heterogeneous localization in intra-I-band structures. In DRG, on the other hand, PIP5Kγ-immunoreactivity was shown to be localized heterogeneously, but not evenly, over apposed plasma membranes of both neurons and ganglionic satellite cells in immune electron microscopy. In addition, no peripheral nerve terminals of DRG showing its distinct immunoreactivity were found in most peripheral fields of nociception and any other sensory perception except for the proprioception through muscle spindles. In contrast, numerous central terminals of DRG in the spinal posterior horn were immunoreactive for it. This finding leads us to consider the possibility that the regulation by PIP5Kγ of nociception is dominantly exerted in DRG and sensory neural tracts central, rather than peripheral, to DRG.     

Stretch receptors in regenerated rat muscle

Although muscle spindles are known to be present in regenerated muscles, it has not previously been reported whether they have any electrophysiological activity. In the present study, rat extensor digitorum longus muscles were traumatized so as to cause all muscle fibers to degenerate; the muscle nerves were either left intact to promote subsequent reinnervation or severed to impede reinnervation. After 2-4 months of regeneration, the muscles were subjected to stretch stimuli and sensory activity was recorded electrophysiologically. Many of the muscles contained stretch-sensitive units that behaved like muscle spindles, although the responses were often highly adaptive and somewhat erratic. In general, the responses from muscles with the nerve left intact were more normal than those from muscles in which the nerve had been severed. Silver staining of the same muscles showed that morphologically recognizable muscle spindles were present, but all were abnormal to varying degrees. These results demonstrate that regenerated muscles can regain some degree of sensory activity in addition to motor functions. The prospect of restored sensory activity may be critical in evaluating the efficacy of graft or transplant procedures for human muscles.     

Muscle spindles in extraocular muscles of human infants

The capacity of muscle spindles in adult human extraocular muscles (EOM) to provide effective proprioception was questioned on the grounds of their peculiar morphology. Their appearance could be attributable to ageing and to test this possibility examples of infant muscle spindles have been examined. Forty encapsulated structures from five extraocular muscles removed post mortem from 4 infant patients aged 6 days, 5, 23 and 30 months were examined by means of light microscopy using serial transverse sections. Seven of them were identified as false spindles. The remaining 33 structures, identified as spindles, contained a total of 175 intrafusal fibres varying from 2 to 12 (mean: 6) in each. 130 of these fibres (74.3%) were of nuclear chain type. Unequivocal evidence of bag fibres was not found. Spindles lacked or had a limited equatorial expansion, and the inner capsule was incomplete and irregularly shaped. 45 (25.7%) of the intrafusal fibres had extrafusal features with large diameters, peripherally placed nuclei, no equatorial modification and without associated sensory nerve terminals. Serial sections revealed that a majority of the nuclear chain fibres were interrupted, fragmented or terminated abruptly, and most spindles contained at least one incomplete fibre. These observations show that the atypical features observed in adult human EOM spindles are also present in infants and are therefore not attributable to ageing.     

Myelin basic protein-positive nerve fibres in human Meissner corpuscles

Myelinated nerve fibres forming sensory corpuscles become amyelinic before entering the corpuscle. Interestingly, in Meissner corpuscles from monkey myelin basic protein (MBP), a specific component of myelin sheath co-localized with neuronal markers. To investigate whether or not this also occurs in human digital Meissner corpuscles, we used single and double immunohistochemistry to detect MBP associated with axonic (protein gene product (PGP) 9.5) or Schwann and Schwann-related cell (S100 protein) markers. We also studied these markers in Pacinian corpuscles. Nerve fibres immunoreactive for MBP were detected in about 25% of the Meissner corpuscles examined; however, MBP never co-localized with PGP 9.5 and MBP occasionally co-localized with S100 protein. MBP-immunoreactive fibres associated with Meissner corpuscles were observed at the periphery of the lamellar cells or within the corpuscle between the lamellar cells. These results describe the distribution of myelinated nerve fibres expressing MBP in human Meissner corpuscles, which is important when studying Meissner corpuscles in cutaneous biopsies used for the diagnosis of peripheral and degenerative neuropathies.     

An ultrastructural study of nerve terminal degeneration in muscle spindles of the tenuissimus muscle of the cat

Summary Muscle spindles in the tenuissimus muscle of the cat were studied between 12 and 168 h after cutting or freezing the nerve to this muscle. Degenerative changes in sensory and motor nerve terminals on intrafusal muscle fibres were observed using the electron microscope. Comparisons were made with spindles from unoperated or sham-operated cats.The earliest degenerative changes were seen in sensory and motor terminals at 20–24 h after the lesion. No nerve endings were seen by 114 h after denervation. The most consistent initial signs of degeneration were: (1) the presence of abnormal mitochondria and dense bodies in sensory terminals, and (2) a decrease in the number and clumping of synaptic vesicles combined with an increase in glycogen and neurofilaments in motor endings. Intrafusal fibres participate in the removal of degenerating sensory endings. Schwann cells phagocytose degenerating motor terminals. The disappearance of nerve terminals precedes the complete degeneration of preterminal myelinated fibres within the muscle spindle.     

Phase contrast synchrotron radiation computed tomography of muscle spindles in the mouse soleus muscle

Muscle spindles are skeletal muscle sensory organs involved in the sensation of position and movement of the body. We have explored the capability of phase contrast computed tomography to visualise muscle spindles in murine skeletal muscle. In particular, we have validated the visualisation of nerve fibres through phase contrast computed tomography using light microscopy on stained histological sections. We further present the first three‐dimensional visualisation of muscle spindles in mouse soleus skeletal muscle in conjunction with the neurovascular bundle associated with it.     

Sensory nerve supply of the human oro‐ and laryngopharynx: A preliminary study

To date, the details of human sensory innervation to the pharynx and upper airway have not been demonstrated. In this study, a single human oro‐ and laryngopharynx obtained from autopsy was processed with a whole‐mount nerve staining technique, Sihler's stain, to determine its entire sensory nerve supply. The Sihler's stain rendered all mucosa and soft tissue translucent while counterstaining nerves. The stained specimen was then dissected and the nerves were traced from their origins to the terminal branches. It was found that the sensory innervation of the human pharynx is organized into discrete primary branches that innervate specific areas, although these areas are often connected by small neural anastomoses. The density of innervation varied, with some areas receiving almost no identifiable nerve supply (e.g., posterior wall of the hypopharynx) and certain areas contained much higher density of sensory nerves: the posterior tonsillar pillars; the laryngeal surface of the epiglottis; and the postcricoid and arytenoid regions. The posterior tonsillar pillar was innervated by a dense plexus formed by the pharyngeal branches of the IX and X nerves. The epiglottis was densely innervated by the internal superior laryngeal nerve (ISLN) and IX nerve. Finally, the arytenoid and postcricoid regions were innervated by the ISLN. The postcricoid region had higher density of innervation than the arytenoid area. The use of the Sihler's stain allowed the entire sensory nerve supply of the pharyngeal areas in a human to be demonstrated for the first time. The areas of dense sensory innervation are the same areas that are known to be the most sensitive for triggering reflex swallowing or glottic protection. The data would be useful for further understanding swallowing reflex and guiding sensory reinnervation of the pharynx to treat neurogenic dysphagia and aspiration disorders. Anat Rec 258:406–420, 2000. © 2000 Wiley‐Liss, Inc.     

The capsule of human Meissner corpuscles: immunohistochemical evidence

Meissner corpuscles are cutaneous mechanoreceptors that are usually located in the dermal papillae of human glabrous skin. Structurally, these sensory corpuscles consist of a mechanoreceptive sensory neuron surrounded by non-myelinating lamellar Schwann-like cells. Some authors have described a partially developed fibroblastic capsule of endoneurial or perineurial origin around Meissner corpuscles; however, others have noted that these structures are non-encapsulated. As there is continuity between the periaxonic cells forming the sensory corpuscles and the cells of the nerve trunks, we used immunohistochemistry to examine the expression of endoneurial (CD34 antigen) or perineurial [Glucose transporter 1 (Glut1)] markers in human cutaneous Meissner corpuscles. We also investigated the immunohistochemical patterns of nestin and vimentin (the main intermediate filaments of the cytoskeleton of endoneurial and perineurial cells, respectively) in Meissner corpuscles. The most important finding from this study was that CD34-positive cells formed a partial/complete capsule of endoneurial origin around most Meissner corpuscles, without signs of other perineurial Glut1-positive elements. However, the cytoskeletal proteins of the capsular CD34-positive cells did not include either nestin or vimentin, so the cytoskeletal composition of these cells remains to be established. Finally, the intensity of the immunoreactivity for CD34 in the capsule decreased with ageing, sometimes becoming completely absent in the oldest individuals. In conclusion, we report the first immunohistochemical evidence of the capsule of Meissner corpuscles in humans and demonstrate the endoneurial origin of the capsule.     

Body ownership and a new proprioceptive role for muscle spindles

Knowledge of which body parts belong to us is referred to as the sense of body ownership. There is increasing evidence that this important aspect of human proprioception is highly malleable. Research into ownership of individual body parts was stimulated by Botvinick and Cohen's rubber‐hand illusion (Nature 391 ,1998, 756), which demonstrated that an artificial body part can be incorporated in one's body representation and can cause real body parts to be sensed erroneously. Here, we review key studies that have advanced our understanding of the sense of body ownership, including the important role played by multisensory integration and spatiotemporal congruence of sensory signals. We also discuss our recent discovery that body ownership can be induced in response to movement stimuli by signals from a single class of sensory receptor, namely muscle spindles.     

Hand fasciae innervation: The palmar aponeurosis

There are few data in the scientific literature about the innervation of fasciae of the hand. The present study first elucidates the density and location of nervous structures in the palmar aponeurosis and, for comparison, in the flexor retinaculum (both can be considered specializations of the deep fascia of the upper limbs). Second, it compares nonpathological with pathological palmar aponeurosis. Samples of nonpathological fascia were taken from the flexor retinaculum and palmar aponeurosis of 16 upper limbs of unembalmed cadavers. Samples of pathological palmar aponeurosis were taken from seven patients with Dupuytren's disease. All samples were stained immunohistochemically with anti‐S100 and anti‐tubulin antibodies, and analyzed quantitatively and qualitatively by microscopy. The palmar aponeurosis showed higher median density than the retinacula of free nerve endings (22 and 20 elements/cm², respectively), Pacinian corpuscles (2 and 0 elements/cm²) and Golgi‐Mazzoni corpuscles (1.0 and 0.5 element/cm²). Some corpuscles were located at the intersections of the fibers in the three directions. Free nerve endings were denser in pathological palmar aponeurosis (38 elements/cm²). The results indicate that the palmar aponeurosis is central to proprioception of the hand and that surgery should therefore avoid injuring it. The higher density of free nerve endings in pathological samples indicates that the nervous structures are implicated in the amplified fibrosis of Dupuytren's disease. Clin. Anat. 31:677–683, 2018. © 2018 Wiley Periodicals, Inc.     

C57BL/6小鼠胫骨前肌和趾长伸肌肌梭解剖学分析

目的 定位C57BL/6小鼠胫骨前肌(TA)和趾长伸肌(EDL)肌梭分布,分析肌梭在骨骼肌中的固定方式,并统计肌梭各区域长度和赤道横截面积(CAS)等参数分析肌梭形态学共性,为肌梭的形态和功能研究提供解剖学基础.方法 5只正常成年C57BL/6小鼠取TA和EDL,利用改良的骨骼肌冷冻技术得到无冰晶样本,样本连续冷冻切片...