Doan H. Nguyen

Clinical Article Accumulation of PN1 and PN3 Sodium Channels in Painful Human Neuroma-Evidence from Immunocytochemistry

Summary.Summary. Background: The axolemmal distribution and density of voltage-gated sodium channels largely determines the electrical excitability of sprouting neurites. Recent evidence suggests that accumulation of sodium channels at injured axonal tips may be responsible for ectopic axonal hyperexcitability and the resulting abnormal sensory phenomena of pain and paresthesias. For future improvement in pain management it is necessary to identify structurally significant generators of autorhythmicity. A first step in this regard will be to determine the predominant types of sodium channels in injured axons. The opportunity to test human specimens from painful and non-painful neuroma is of great value. Methods: We employed immunocytochemical methods to investigate if two types of highly specific voltage-gated sodium channel subtypes could be detected in sections of human neuroma. Findings: Both subtypes of sodium channels PN1 and PN3 accumulated abnormally in human neuromas. The immunoreactive pattern was more pronounced in painful neuromas. This is in contrast to previous reports that focused either on PN1 or PN3 as main generators of hyperexcitability induced pain. Interpretation: Both, PN1 and PN3 seem to be involved in hyperexcitability induced pain. It can be expected that a variety of other highly specific voltage gated sodium channel subtypes will be detected in regenerating peripheral nerve in the near future, which contribute to the development of neuropathic pain states. Thus, in order to therapeutically control hyperexcitability induced neuropathic pain, it might be worthwhile to develop pharmaceuticals that can selectively block different sodium channel subtypes and subunits. A review of the role of sodium channels in neuropathic pain is implemented in the discussion.

Tumor necrosis factor-alpha and interleukin-1 induce activation of MAP kinase and SAP kinase in human neuroma fibroblasts.

Two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), which are released by macrophages during the early inflammatory phase of nerve injury, are known to induce activation of mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK), which locate at different signal transduction pathways and are involved in cell cycle G0/G1 transition and cellular proliferation in human fibroblasts. Activation of these two protein kinases by the cytokines may stimulate fibroblast proliferation in damaged nerves and thereby play a role in the formation of a neuroma, a disorganized mass of tissue that interferes with neural regeneration and repair. To investigate the possibility that this mechanism is operative in neuroma formation, we used cultured, serum-starved fibroblasts from surgically removed human neuromas stimulated with TNF-alpha and/or IL-1 alpha and IL-1 beta, and measured the activation of MAPK and SAPK using myelin basic protein (MBP) and human c-Jun (1-169) glutathione S-agarose transferase (GST) fusion protein as substrates. For comparison, neuroma fibroblast cultures were also stimulated with phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor-AB (PDGF-AB), a potent activator for MAPK. TNF-alpha and both forms of IL-1 produced a rapid activation of MAPK, with a peak at 15 min for TNF-alpha stimulation, and a peak at 30 min for IL-1 stimulation. TNF-alpha combined with either IL-1 alpha or IL-1 beta produced a synergistic effect on the activation of MAPK. The increases in MAPK induced by TNF-alpha and IL-1 were similar to the increases induced by PMA and PDGF-AB. To confirm the presence of MAPK, immunoprecipitation and immunoblotting were carried out on experimental and control lysates. TNF-alpha and IL-1 also increased activation of SAPK, but to a lesser extent than MAPK. PMA and PDGF-AB were also much less effective in stimulating activation of SAPK. Our findings indicate that TNF-alpha and IL-1 activate parallel signal transduction pathways in human neuroma fibroblasts, and that they are relatively stronger activators of MAPK than of SAPK. Previous studies have convincingly demonstrated that MAPK and SAPK are involved in human fibroblast proliferation. The results of our study suggest that TNF-alpha and IL-1 may play a role in frustrating functional nerve regeneration after injury by stimulating these two kinases, which, in turn, leads to fibroblast proliferation and formation of neuromas.

EFFECT OF SENSORY DENERVATION ON THE STRUCTURE AND PHYSIOLOGIC RESPONSIVENESS OF RABBIT LACRIMAL GLAND

Purpose This work was conducted to determine the effects of unilateral trigeminal ganglion ablation on lacrimal gland structure and secretory activity. Methods Adult male New Zealand rabbits underwent unilateral thermocoagulation of the ophthalmic division of the trigeminal ganglion. Sensory denervation was affirmed by anatomic inspection of the lesion and transmission electron microscopy (TEM) of the lacrimal gland innervation. Eight to 10 days after the procedure, the intraorbital lacrimal glands were removed from both sides. To compare the physiologic competence of the intact and denervated glands, freshly isolated gland fragments from the paired intact and denervated glands were stimulated with carbachol (100 μM), isoproterenol (10 μM), phorbol-12,13-dibutyrate (PDBu, 10 μM), forskolin (40 μM), or vehicle. Total secreted protein was measured at 30 or 60 min after the establishment of baseline values. Intact and denervated glands also were examined by light and TEM, and the morphologic appearance of the acinar structures as well as the appearance of nerves innervating the gland after denervation were assessed. Similar experiments were conducted with animals that underwent unilateral superior cervical ganglionectomy. Results Tissues from sensory denervated glands released significantly more protein than did tissues from innervated glands in response to in vitro stimulation by carbachol or isoproterenol but not in response to PDBu or forskolin. Microscopy showed that the acinar cells that had undergone sensory denervation showed a massive accumulation of secretory granules. The secretory granules filled the entire cytoplasmic space and displaced the ellipsoidal nuclei to the extreme periphery. Examination of segments of nerves revealed numerous unmyelinated axons, a few small-diameter myelinated axons, and a large amount of nerve degeneration after sensory denervation. In contrast to the effects of sensory denervation, sympathetic denervation did not alter either the acinar appearance or secretory responsiveness of the gland. Conclusion Loss of the considerable sensory innervation from the trigeminal ganglion has pronounced effects on the pharmacologic responsiveness and the structure of the lacrimal gland. The effects of sensory innervation on the gland may be mediated through two possible pathways: direct input to the gland or control of the preganglionic parasympathetic pathway.

GROWTH FACTOR AND NEUROTROPHIC FACTOR MRNA IN HUMAN LACRIMAL GLAND

Purpose This study examined the expression of important growth factor and receptor messenger RNA (mRNA) in human lacrimal gland. Method Lacrimal gland tissue was obtained from six women, aged 31–85 years, who were undergoing surgery. The specimens were frozen immediately in liquid nitrogen. Total cellular RNA was collected by cesium chloride centrifugation, and the integrity of the RNA was analyzed by gel electrophoresis and spectrophotometry. For the reverse-transcription polymerase chain reaction (RT-PCR) procedure, I μg of total cellular RNA was used for the first strand synthesis and amplified in separate reactions for 34 cycles by using primers specific for transforming growth factor-β 1,-β 2, and -β 3 (TGFβ1, TGFβ2, TGFβ3), TGFβ3 receptor (TGFβ3R), basic fibroblast growth factor (bFGF), fibroblast growth factor receptor-1 (FGF-RI), nerve growth factor β-NGF), low-affinity NGF receptor (p75NGF-R), and platelet-derived growth factor-AA (PDGF-AA; two of the six cases) and -BB (PDGF-BB; three of the six cases). Product identity was confirmed by restriction endonuclease digestion, by the “hot blot” technique, by DNA sequencing, or by a combination of these. Results All the lacrimal gland specimens were positive for all growth factors, neurotrophic factors, and receptors except for two specimens that were negative for bFGF. The patients from whom the bFGF-negative specimens were obtained were 68 and 74 years old, and both were identified as having dry eyes—one severe and the other moderate, respectively. These specimens were also analyzed for PDGF-AA and PDGF-BB mRNA, and both were positive. Conclusions This study demonstrates that RT-PCR is a rapid and sensitive method for analyzing rare mRNA transcripts in small amounts of tissue. The results suggest that these growth and neurotrophic factors may have autocrine and paracrine roles in modulating the lacrimal gland, and their absence may play a role in pathologic states such as fibrosis and dry eyes.

CHARACTERIZATION OF IMMORTALIZED RABBIT LACRIMAL GLAND EPITHELIAL CELLS

SummaryTo establish an immortalized lacrimal gland epithelial cell line, the orbital lacrimal glands of normal New Zealand White rabbits were multiply injected with an immortalizing amphotropic retroviral vector (LXSN16E6E7) containing the E6 and E7 genes of human papillomavirus type 16. Lacrimal glands were removed after 2 d and acinar epithelial cells were isolated and cultured on Matrigel-coated 60 mm2 plates containing DMEM-F12 supplemented with 5% Nu-serum V. Transformed cells were selected in G418 sulfate for 7 d and passaged. Morphology of the immortalized cells was similar to that described for normal acinar cells both in vivo and in vitro, with rough endoplasmic reticulum and secretory granules. These characteristics remained unchanged and the cells continued to exhibit typical polygonal epithelioid structure. The cells have been maintained in culture for 14 mo. and have gone through 58 passages without loss of proliferation or epithelial cell characteristics. Immunohistochemistry and Western blots showed positive reactivity to secretory component, transferrin, and transferrin receptor, which are typical proteins found in the lacrimal gland. Functional analysis by stimulation with a cholinergic agonist, carbachol (100 µM), resulted in a significant release of protein. This is the first report of an immortalized rabbit lacrimal epithelial cell. These cells will provide a valuable tool for the molecular analysis of lacrimal gland epithelial cell functions.

Ankyrin G and voltage gated sodium channels colocalize in human neuroma – key proteins of membrane remodeling after axonal injury

We tested if ankyrin G could be detected in human neuroma, if it colocalized with site-specific peripheral nerve sodium channels that accumulate at axon tips of injured nerve, and if there are differences in the distribution of these proteins in non-painful neuroma and painful neuroma tissue vs. normal nerve. Frozen sections from one painful, six non-painful, and three normal nerves were immunocytochemically examined. A double labeling technique with highly specific antibodies against peripheral nerve type 1 (Na(v)1.7), and peripheral nerve type 3 (Na(v)1.8) sodium channels and anti-ankyrin G antibodies detected sodium channels and ankyrin G on the same section, using confocal laser scanning microscopy. Ankyrin G colocalized with both types of sodium channels. Neuroma specimens exhibited considerably larger immunofluorescence for both sodium channels and ankyrin G compared with normal nerve. The painful neuroma presented an even more pronounced immunolabeling in clusters. Findings support results from animal models that link ankyrin G with clustering of sodium channels at axon tips of unmyelinated, sprouting fibers. A common (repair-) mechanism that exists throughout the human nervous system for clustering sodium channels at a high density is assumed. A dysregulation in this membrane remodeling mechanism might be an initial step in a cascade that leads to a painful rather than a non-painful neuroma.

Neurologic Evaluation of Acute Lacrimomimetic Effect of Cyclosporine in an Experimental Rabbit Dry Eye Model

PURPOSE To evaluate neurologically acute lacrimation caused by cyclosporine (CsA) eyedrops in rabbit. METHODS Normal adult male New Zealand White rabbits and those that underwent parasympathectomy each received a single instillation of 0.1% CsA or vehicle eyedrops. Schirmer tear test (STT) results, flow rate of lacrimal gland (LG) fluid from the excretory lacrimal duct of the main LG, and blink rate (over a 3-minute period) were measured before and after instillation of CsA or vehicle. Light microscopy was performed to examine the main LG in vitro. Protein release from LG fragments was assessed after incubation with CsA for 30 minutes. RESULTS In normal rabbits, the STT value and the flow rate of LG fluid were significantly increased after treatment with CsA compared with vehicle (P < 0.05). In contrast, no changes were found in denervated eyes. The blink rate of CsA-treated eyes was significantly higher than that of vehicle-treated eyes in normal rabbits (P < 0.005), whereas that of denervated eyes decreased significantly after CsA instillation compared with before administration (P < 0.005). Light microscopy showed that the cytoplasm of acinar cells was packed with secretory granules in denervated LG tissue 7 days after parasympathectomy. The same finding was observed 3 hours after CsA instillation. CsA had no stimulatory effect on protein release by acinar cells in LG fragments at all concentrations tested. CONCLUSIONS These results suggest that CsA has no direct effect on tear fluid secretion from the LG in an acute model. Instead, CsA increases reflex tear flow.

Sensory Denervation Leads to Deregulated Protein Synthesis in the Lacrimal Gland

The secretory function of the lacrimal gland is influenced by the release of classic and peptide neurotransmitters from sensory, sympathetic, and parasympathetic nerve terminals in the gland. These neuronal pathways constitute an integrated system that regulates the integrity of the front of the eye, particularly the optical qualities of the cornea. In everyday experience, this relationship is evidenced by the rapid tearing response to corneal stimulation. The release of neuromodulators resulting from activation of these pathways leads to receptor activation, mobilization of intracellular second messengers, exocytosis, and synthesis of secretory proteins. Consequently, the processes of cell signaling and synthesis of new secretory material must be regulated in coordination with secretion to maintain the normal exocrine function of the gland.

Transcription of message for tumor necrosis factor-alpha by lacrimal gland is regulated by corneal wounding.

Tumor necrosis factor-alpha (TNF), or cachectin, has been implicated in the pathophysiology of wasting diseases and inflammation.1 Although most cells have receptors for this hormone, the receptor-mediated events may range from a promotion of fibrosis to cell death.1,2 The properties of TNF suggest that it could play a role in the progress of lacrimal gland disease, such as Sjogren’s syndrome, in which fibrosis and lymphocytic infiltration are both present.3

Loss of parasympathetic innervation leads to sustained expression of pro-inflammatory genes in the rat lacrimal gland.

It has been shown that removal of parasympathetic innervation to the lacrimal gland (LG) leads to rapid reduction in tear flow. Additionally, removal of the neural input resulted in disorganization of LG structure and changes in the expression of genes associated with the secretory pathway and inflammation. The goal of this study was to investigate the change in pro-inflammatory and pro-apoptotic gene expression in the rat LG following parasympathetic denervation. Male Long-Evans rats underwent unilateral sectioning of the greater superficial petrosal nerve and were sacrificed 7 days or 2.5 months later. cDNA was synthesized from LG RNA from the contralateral control (Ctla) and parasympathectomized (Px) glands and comparative real-time PCR was performed. Mean threshold cycles (MC(T)) for the Ctla and Px LG genes were normalized to 18S rRNA MC(T) values, and the relative fold change was calculated for each gene using the 2(-DeltaDeltaC)(T) method. The expression of nuclear factor kappa B1, caspase 1, eotaxin, leukocyte antigen MRC-OX44, allograft inflammatory factor-1, MHC class II molecules RT.1B and RT.1D, IgG receptor FcRn, and macrophage metalloelastase was increased and remained elevated in the Px LG, compared with the Ctla LG. Increased expression of the initiator of apoptosis gene, caspase 2, was confirmed, but expression of the executor gene, caspase 6, was not elevated in the Px LG. Reduced expression of genes associated with post-translational protein processing-furin convertase, protein disulfide isomerase, and UDP-gal transporter isozyme 1-was noted in the Px LG. No significant changes in the expression of genes associated with lysosomal and non-lysosomal-mediated protein degradation were found. Removal of parasympathetic input may lead to decreased capacity for protein synthesis and elevated immune responses in the Px LG. These changes occur without increases in expression of the muscarinic acetylcholine receptor subtype 3, and may suggest the early changes in LG acinar cells and the pathophysiology of autoimmune responses.