Transforming growth factor beta 1, a cytokine with regenerative functions.

We review the biology and role of transforming growth factor beta 1 (TGF-ß1) in peripheral nerve injury and regeneration, as it relates to injuries to large nerve trunks (i.e., sciatic nerve, brachial plexus), which often leads to suboptimal functional recovery. Experimental studies have suggested that the reason for the lack of functional recovery resides in the lack of sufficient mature axons reaching their targets, which is a result of the loss of the growth-supportive environment provided by the Schwann cells in the distal stump of injured nerves. Using an established chronic nerve injury and delayed repair animal model that accurately mimics chronic nerve injuries in humans, we summarize our key findings as well as others to better understand the pathophysiology of poor functional recovery. We demonstrated that 6 month TGF-ß1 treatment for chronic nerve injury significantly improved Schwann cell capacity to support axonal regeneration. When combined with forskolin, the effect was additive, as evidenced by a near doubling of regenerated axons proximal to the repair site. We showed that in vivo application of TGF-ß1 and forskolin directly onto chronically injured nerves reactivated chronically denervated Schwann cells, induced their proliferation, and upregulated the expression of regeneration-associated proteins. The effect of TGF-ß1 and forskolin on old nerve injuries is quite impressive and the treatment regiment appears to mediate a growth-supportive milieu in the injured peripheral nerves. In summary, TGF-ß1 and forskolin treatment reactivates chronically denervated Schwann cells and could potentially be used to extend and prolong the regenerative responses to promote axonal regeneration.

Ethanol upregulates glucocorticoid-induced leucine zipper expression and modulates cellular inflammatory responses in lung epithelial cells.

Alcohol abuse is associated with immunosuppressive and infectious sequelae. Particularly, alcoholics are more susceptible to pulmonary infections. In this report, gene transcriptional profiles of primary human airway epithelial cells exposed to varying doses of alcohol (0, 50, and 100 mM) were obtained. Comparison of gene transcription levels in 0 mM alcohol treatments with those in 50 mM alcohol treatments resulted in 2 genes being upregulated and 16 genes downregulated by at least 2-fold. Moreover, 0 mM and 100 mM alcohol exposure led to the upregulation of 14 genes and downregulation of 157 genes. Among the upregulated genes, glucocorticoid-induced leucine zipper (GILZ) responded to alcohol in a dose-dependent manner. Moreover, GILZ protein levels also correlated with this transcriptional pattern. Lentiviral expression of GILZ small interfering RNA in human airway epithelial cells diminished the alcohol-induced upregulation, confirming that GILZ is indeed an alcohol-responsive gene. Gene silencing of GILZ in A549 cells resulted in secretion of significantly higher amounts of inflammatory cytokines in response to IL-1beta stimulation. The GILZ-silenced cells were more resistant to alcohol-mediated suppression of cytokine secretion. Further data demonstrated that the glucocorticoid receptor is involved in the regulation of GILZ by alcohol. Because GILZ is a key glucocorticoid-responsive factor mediating the anti-inflammatory and immunosuppressive actions of steroids, we propose that similar signaling pathways may play a role in the anti-inflammatory and immunosuppressive effects of alcohol.

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.

Evaluation of novel dry eye model: preganglionic parasympathetic denervation in rabbit.

PURPOSE: To evaluate ocular surface status after interruption of preganglionic, parasympathetic neural control after surgical removal of the greater superficial petrosal nerve (GSPN). METHODS: New Zealand White rabbits underwent unilateral section and removal of a 5-mm portion of the GSPN by a route through the inner ear; no ocular or orbital tissue was involved. Before and 7 days after surgery, all animals underwent preliminary examination, including fluorescein staining, rose bengal instillation, blink rate, tear breakup time (BUT), tear flow, and impression cytology. Total tarsorrhaphy was carried out in four additional rabbits, and another four animals underwent unilateral sham procedures. The GSPN, pterygopalatine ganglion, lacrimal gland, and conjunctiva were evaluated by light and transmission electron microscopy (TEM). RESULTS: GSPN sectioning resulted in significant changes of the ocular surface after 7 days: intense rose bengal staining of the conjunctiva, fluorescein staining of the cornea, increased blink rate (P < 0.05), decreased BUT (P < 0.005), decreased tear flow by 26% (P < 0.005), and decreased goblet cell density (P < 0.01). TEM revealed massive accumulation of secretory granules in lacrimal acinar cells. The changes were also seen after tarsorrhaphy. Neither the contralateral control nor the sham eyes were affected. CONCLUSIONS: The effects of GSPN nerve section led to the rapid onset of a dry eye condition in the rabbits that continued for at least 1 week. The authors suggest that continuous neural drive of the pterygopalatine ganglion is necessary to maintain adequate tear flow and mucin secretion. It is likely the trigeminal system is the afferent origin of this continuous neural tone.

Sensory denervation modulates eIF-2 alpha kinase expression in the rabbit lacrimal gland.

PURPOSE: To investigate the hypothesis that sensory denervation of the rabbit lacrimal gland results in dysregulation of protein synthesis. We used differential display of mRNA to identify genes associated with protein synthesis and secretion that may be altered in this situation. METHODS: New Zealand white rabbits underwent unilateral sensory denervation by the ablation of the trigeminal ganglion. After 7 days, the denervated and contralateral control lacrimal glands were removed. The effects of denervation on gene expression were carried out using differential mRNA display. Northern and Western blot analyses were used to verify differential gene expression. RESULTS: Differential mRNA display identified the gene heme-regulated inhibitor eukaryotic initiation factor-2 alpha kinase (HRI eIF-2a kinase) in the lacrimal gland, the expression of which was reduced in the denervated lacrimal gland. The sequenced fragment from differential display showed 94% identity to rabbit HRI eIF-2a kinase. The decreased expression of HRI eIF-2a kinase was confirmed by Northern and Western blots, and measurement of HRI eIF-2a kinase phosphorylation activity in the lacrimal gland after ablation of sensory neurons showed that it was significantly decreased compared with that of normal and control lacrimal glands. CONCLUSIONS: The results suggest that loss of sensory innervation has a role in the lacrimal gland, contributing to the expression of HRI eIF-2a kinase, a pivotal negative regulator of protein synthesis. A reduction in control of protein synthesis may lead to the translation of repressed messages associated with cell stress responses.

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.

Elevated ankyrin G in a plexiform neurofibroma and neuromas associated with pain.

UNLABELLED: Ankyrin G has recently been shown to be responsible for activation of sodium channels in the developing and regenerating axonal membrane. Via this sodium channel mechanism, elevated ankyrin G levels have been linked with spontaneous ectopic hyperexcitability and thus with pain phenomena in nervous tissue. Ankyrin G, a transmembrane, structural protein of the axon, was examined in four conditions: (a) painful plexiform neurofibroma; (b) painful neuroma; (c) non-painful neuromas; (d) normal nerve. Neurofibroma tissue was obtained from an 18-year old male patient who developed an intensely painful, plexiform neurofibroma of the posterior femoral cutaneous nerve and subsequently underwent surgery. Sample proteins were separated by PAGE and labeled with anti-ankyrin G antibodies in a Western blot procedure. RESULTS: The ankyrin G band density (mug) of protein for the painful neurofibroma was 6014 and was 3557 for the painful neuroma as compared to 3041, 1988 and 606 (mean+/-SD=1878+/-1221) for the three non-painful neuromas. Ankyrin G expression in normal nerves (8 specimens from 7 patients) was comparatively less (mean+/-SD=411+/-339). CONCLUSION: Our results may represent the first evidence for abnormally increased levels of ankyrin G protein with painful neurofibromas. Due to ankyrin G's multifunctional role in the development and remodeling of excitable membranes, it can be hypothesized that the significant increase contributes to the development of hyperexcitable axonal membranes in neurofibromas and potentially other peripheral pain conditions.

Microarray analysis of the rat lacrimal gland following the loss of parasympathetic control of secretion.

Previous studies showed that loss of muscarinic parasympathetic input to the lacrimal gland (LG) leads to a dramatic reduction in tear secretion and profound changes to LG structure. In this study, we used DNA microarrays to examine the regulation of the gene expression of the genes for secretory function and organization of the LG. Long-Evans rats anesthetized with a mixture of ketamine/xylazine (80:10 mg/kg) underwent unilateral sectioning of the greater superficial petrosal nerve, the input to the pterygopalatine ganglion. After 7 days, tear secretion was measured, the animals were killed, and structural changes in the LG were examined by light microscopy. Total RNA from control and experimental LGs (n = 5) was used for DNA microarray analysis employing the U34A GeneChip. Three statistical algorithms (detection, change call, and signal log ratio) were used to determine differential gene expression using the Microarray Suite (5.0) and Data Mining Tools (3.0). Tear secretion was significantly reduced and corneal ulcers developed in all experimental eyes. Light microscopy showed breakdown of the acinar structure of the LG. DNA microarray analysis showed downregulation of genes associated with the endoplasmic reticulum and Golgi, including genes involved in protein folding and processing. Conversely, transcripts for cytoskeleton and extracellular matrix components, inflammation, and apoptosis were upregulated. The number of significantly upregulated genes (116) was substantially greater than the number of downregulated genes (49). Removal of the main secretory input to the rat LG resulted in clinical symptoms associated with severe dry eye. Components of the secretory pathway were negatively affected, and the increase in cell proliferation and inflammation may lead to loss of organization in the parasympathectomized lacrimal gland.

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.

Painful neuromas: a potential role for a structural transmembrane protein, ankyrin G.

OBJECT: Severe nerve injury induces the formation of a neuroma. Some neuromas cause excruciating pain. Overexpression of Na+ channels leads to hyperexcitability and painful phenomena. Ankyrin G, a multifunctional transmembrane protein of the axolemma, might be a key protein in neuroma formation because it binds Na+ channels in the initial segments of a regenerating axon and links with neuronal cell adhesion molecules. The authors wanted to determine if ankyrin G could be detected in neuroma, and if present, whether there would be differences in distribution between nonpainful neuromas, painful neuromas, and normal nerve. METHODS: First, frozen sections of nine nerve specimens obtained from six patients (six nonpainful neuromas, one painful neuroma, and two normal nerves) were immunocytochemically screened for ankyrin G by using confocal laser scanning microscopy. Second, specimens from 29 patients (seven painful neuromas, 15 nonpainful neuromas, and seven normal nerves) were examined using immunoblot analysis for their ankyrin G content. Western blot analysis detected ankyrin G, which was visualized by applying the enhanced chemiluminescence technique. Computerized densitometry was used to quantitate ankyrin G expression by comparing band intensities. Normal nerve served as control. Neurofilament was used as a marker for nerve tissue content. Ankyrin G could be detected and was found to be increased in neuromas. The mean band intensity values were 1838 for painful neuromas, 1166 for nonpainful neuromas, and 411 for normal nerves. In two cases the authors were able to compare specimens of painful neuroma and normal nerve from the same patient. The painful neuromas exhibited considerably higher levels of ankyrin G. Painful neuroma and normal nerve densitometry values were 499 and 165, respectively, for one patient, and 4254 and 821, respectively, for the other patient. Painful neuromas were also found to have higher neurofilament values than nonpainful neuromas. CONCLUSIONS: Altered regulation of ankyrin G after nerve injury may lead to hyperexcitability and painful phenomena via clustering of Na+ channels. A propensity to overexpress ankyrin G after peripheral nerve trauma may turn out to be a factor in the development of painful neuromas and neuropathic pain. The relevant literature regarding the importance of ankyrin G for nerve regeneration and nerve membrane remodeling is reviewed.