M. E. Del Valle

Immunohistochemical analysis of mechanoreceptors in the human posterior cruciate ligament

Although long-term studies report successful results with total knee arthroplasty (TKA), performed with or without posterior cruciate ligament (PCL) retention, controversy exists as to which is preferable in regard to patient outcome and satisfaction. The possible proprioceptive role of the PCL may account for a more normal feeling of the arthroplasty. Although the PCL has been examined using various histological techniques, immunohistochemical techniques are the most sensitive for neural elements. Therefore an immunohistochemical study was designed to determine the patterns of innervation, the morphological types of the proprioceptors, and their immunohistochemical profile. During TKA, samples were obtained from 22 osteoarthritic PCLs and subjected to immunohistochemical analysis with mouse monoclonal antibodies against neurofilament protein (NFP), S100 protein (S100P), epithelial membrane antigen (EMA), and vimentin (all present in neuromechanoreceptors). Three normal PCLs from cadaveric specimens were also obtained and analyzed for comparison. Five types of sensory corpuscles were observed in both the normal and the arthritic PCLs: simple lamellar, Pacini-like, Ruffini, Krause-like, and morphologically unclassified. Their structure included a central axon, inner core, and capsule in lamellar and Pacini corpuscles and variable intracorpuscular axons and periaxonal cells in the Ruffini and Krause-like corpuscles. The immunohistochemical profile showed the central axon to have NFP immunoreactivity, periaxonal cells to have S100P and vimentin immunoreactivity, and the capsule to have EMA and vimentin immunoreactivity. Nerve fibers and free nerve endings displayed NFP and S100P immunoreactivity. The immunohistochemical profile of the PCL sensory corpuscles is almost identical to that of cutaneous sensory corpuscles. Some prior histological studies of the PCL reported Golgi-like mechanoreceptors, and others found encapsulated corpuscles but no Golgi-like structures. This report determined the innervation of the PCL by the more sensitive immunohistochemical means, revealing four major types of encapsulated mechanoreceptors. The plentiful and varied types of encapsulated mechanoreceptors found in even the arthritic PCL suggests a rich proprioceptive role. It is controversial as to whether preservation of the PCL at TKA improves postoperative proprioception. Our findings tend to support those clinical reports of improved proprioception after PCL-retaining versus PCL-substituting TKAs. The presence of many and varied types of mechanoreceptors may account for the improved stair climbing reported in patients with PCL-retaining TKA and may contribute to patient satisfaction and a more normal feeling after TKA.

Immunohistochemistry of human cutaneous Meissner and Pacinian corpuscles

This paper reviews the immunohistochemical characteristics of two kinds of human cutaneous sensory nerve formations (SNFs), the Meissner and Pacinian corpuscles. In both kinds of SNF the central axon might be easily identifiable because it displays immunoreactivity (IR) for the neuroendocrine markers neuron‐specific enolase and protein gene product 9.5, as well as for neuron‐specific intermediate filament proteins, i.e., neurofilaments. Other intermediate filament proteins such as vimentin are localized in the lamellar cells of Meissner corpuscles, and in the inner core, outer core and capsule of Pacinian corpuscles. However, they lack cytokeratins or glial fibrillary acidic protein IR. On the other hand, and in agreement with ultrastructural data, IR for basement membrane constituents laminin and type IV collagen is found underlying all SNF constituents, with the exception of the axon.

The Trk tyrosine kinase inhibitor K252a regulates growth of lung adenocarcinomas

The neurotrophin family of growth factors and their receptors support the survival of several neuronal and non-neuronal cell populations during embryonic development and adult life. Neurotrophins are also involved in malignant transformation. To seek the role of neurotrophin signaling in human lung cancer we studied the expression of neurotrophin receptors in human lung adenocarcinomas and investigated the effect of the neurotrophin receptor inhibitor K252a in A549 cell survival and colony formation ability in soft agar. We showed that human lung adenocarcinomas express TrkA and TrkB, but not TrkC; A549 cells, derived from a human lung adenocarcinoma, express mRNA transcripts encoding nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), TrkA, TrkB, and p75, and high protein levels of TrkA and TrkB. Stimulation of cells using NGF or BDNF activates the anti-apoptotic protein Akt. Interestingly, inhibition of neurotrophin receptor signaling using K252a prevents Akt activation in response to NGF or BDNF, induces apoptotic cell death, and diminishes the ability of A549 cells to growth in soft agar. The data suggest that neurotrophin signaling inhibition using k252a may be a valid therapy to treat patients with lung adenocarcinomas.

S100 protein-like immunoreactivity in the crypt olfactory neurons of the adult zebrafish

The olfactory epithelium of some teleosts, including zebrafish, contains three types of olfactory sensory neurons. Because zebrafish has become an ideal model for the study of neurogenesis in the olfactory system, it is of capital importance the identification of specific markers for different neuronal populations. In this study we used immunohistochemistry to analyze the distribution of S100 protein-like in the adult zebrafish olfactory epithelium. Surprisingly, specific S100 protein-like immunostaining was detected exclusively in crypt neurons, whereas ciliated and microvillous neurons were not reactive, and the supporting glial cells as well. The pattern of immunostaining was exclusively cytoplasmic without apparent polarity within the soma, and the intensity of immunostaining was not related with the maturative stage of the neurons. The role of S100 protein in crypt olfactory neurons is unknown, although it is probably associated with the capacity of these cells to respond to chemical stimuli. In any case, it represents an excellent marker to identify crypt olfactory neurons in zebrafish.

Characterization of sensory deficits in TrkB knockout mice

The sensory deficit in TrkB deficient mice was evaluated by counting the neuronal loss in lumbar dorsal root ganglia (DRG), the absence of sensory receptors (cutaneous--associated to the hairy and glabrous skin - muscular and articular), and the percentage and size of the neurocalcin-positive DRG neurons (a calcium-binding protein which labels proprioceptive and mechanoceptive neurons). Mice lacking TrkB lost 32% of neurons, corresponding to the intermediate-sized and neurocalcin-positive ones. This neuronal lost was accomplished by the absence of Meissner corpuscles, and reduction of hair follicle-associated sensory nerve endings and Merkel cells. The mutation was without effect on Pacinian corpuscles, Golgis organs and muscle spindles. Present results further characterize the sensory deficit of the TrkB-/- mice demonstrating that the intermediate-sized neurons in lumbar DRG, as well as the cutaneous rapidly and slowly adapting sensory receptors connected to them, are under the control of TrkB for survival and differentiation. This study might serve as a baseline for future studies in experimentally induced neuropathies affecting TrkB positive DRG neurons and their peripheral targets, and to use TrkB ligands in the treatment of neuropathies in which cutaneous mechanoreceptors are primarily involved.

Mechanosensory neurons, cutaneous mechanoreceptors, and putative mechanoproteins

The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid‐sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock‐out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited. Microsc. Res. Tech. 75:1033–1043, 2012.

Differential Localization of Acid-Sensing Ion Channels 1 and 2 in Human Cutaneus Pacinian Corpuscles

Acid-sensing ion channels (ASICs) are the members of the degenerin/epithelial sodium channel (Deg/ENaC) superfamily which mediate different sensory modalities including mechanosensation. ASICs have been detected in mechanosensory neurons as well as in peripheral mechanoreceptors. We now investigated the distribution of ASIC1, ASIC2, and ASIC3 proteins in human cutaneous Pacinian corpuscles using immunohistochemistry and laser confocal-scanner microscopy. We detected different patterns of expression of these proteins within Pacinian corpuscles. ASIC1 was detected in the central axon co-expressed with RT-97 protein, ASIC2 was expressed by the lamellar cells of the inner core co-localized with S100 protein, and ASIC3 was absent. These results demonstrate for the first time the differential distribution of ASIC1 and ASIC2 in human rapidly adapting low-threshold mechanoreceptors, and suggest specific roles of both proteins in mechanotransduction.

Calbindin D-28k immunoreactivity in the rat cerebellar cortex: age-related changes

The present study was designed to analyze age-dependent changes in the expression of calbindin D-28k immunoreactivity in the cerebellar cortex of male Wistar rats aged 3 months (young), 12 months (adult) and 24 months (old). Calbindin D-28k immunoreactivity was localized primarily in the cytoplasm of Purkinje neurons and in the basal portion of their dendritic processes entering in the molecular layer. The expression of calbindin D-28k immunoreactivity was highest in Purkinje neurons of adult rats and lowest in Purkinje neurons of old rats. Moreover, the number of Purkinje neurons displaying calbindin D-28k immunoreactivity was decreased in aged rats. These results demonstrate the occurrence of age-related-changes in the expression of immunoreactivity of the Ca2+ binding protein calbindin D-28k in the rat cerebellar cortex. The possibility that impairment in the expression of Ca2+ binding proteins may be involved in changes of intracellular Ca2+ homeostasis reported in aging and in some neuro-degenerative disorders is discussed.

Neurotrophin receptor proteins immunoreactivity in human gastrointestinal endocrine cells

The distribution of neurotrophin receptors (p75, trkA-, trkB-, and trkC-receptor proteins) was studied by immunohistochemistry on sections of human gastrointestinal tract mucosa from esophagus through rectum. Moreover, chromogranin A (CgA) was studied in parallel to identify endocrine cells (EC). In all of the analyzed samples there was specific immunoreactivity (IR) for trkB-receptor protein in EC, the percentage of which varied between 26 +/- 0.6% for the duodenum and 78 +/- 3% for the sigmoid colon. EC displaying trkC-receptor protein IR were also encountered, in some cases, in EC of the gastric fundus (9%), duodenum (12%), jejune (23%), and colon (12%); trkA-receptor protein IR was occasionally present labelling EC in the jejune (52%), ileum (25%), and sigmoid colon (18%); finally, p75 was in 21% of EC exclusively in one case in the ileum. In addition to EC, IR for all assessed antigens was also present in the submucous blood vessels. Our results provide evidence for the occurrence of neurotrophin receptor proteins in nonneuronal tissues and suggest that neurotrophins, especially that binding trkB receptor proteins, can regulate a subpopulation of EC cells. However, whether EC expressing different trk receptor proteins represent neurochemical subtypes of EC, and whether the identified trk receptor proteins correspond to functional receptors, remain to be elucidated.

Immunoreactivity for phosphorylated 200-kDa neurofilament subunit is heterogeneously expressed in human sympathetic and primary sensory neurons.

This study was undertaken to investigate whether human sensory and sympathetic neurons contain phosphorylated neurofilament proteins, and whether they may be classified on the basis of this property, as in other mammalian species. The distribution of the phosphorylated 200-kDa neurofilament protein subunit (p200-NFP) was investigated in lumbar sympathetic and dorsal root ganglia by means of the RT97 monoclonal antibody (against p200-NFP). The intensity of immunostaining, and the size of neuronal body profiles were measured in order to define different neuron subclasses. In dorsal root ganglia, most of the neuronal profiles (96%) were p200-NFP immunoreactive, and the intensity of immunostaining was not related to neuronal perikarya size. In the lumbar paravertebral sympathetic ganglia, virtually all neurons displayed p200-NFP immunoreactivity, and the intensity of immunolabelling was also independent of the size of the neuronal somata. These results demonstrate heterogeneity in the expression of p200-NFP immunoreactivity in human sympathetic and sensory neurons. In contrast to other mammalian species, RT97 immunolabelling cannot be used as a discriminative marker for the two main types of human primary sensory neurons. On the other hand, our findings provide evidence for the occurrence of phosphorylated neurofilaments within peripheral neuron cell bodies.