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Nerve growth factor and corneal wound healing in dogs
Experimental Eye Research 80 (2005) 633–642 www.elsevier.com/locate/yexer
Nerve growth factor and corneal wound healing in dogs Heung-Myong Wooa,1, Ellison Bentleya, Sean F. Campbella, Carl F. Marfurtb, Christopher J. Murphya,* a
Department of Surgical Sciences, School of Veterinary Medicine, School of Medicine, University of Wisconsin-Madison, 2015 Linden Drive W, Madison, WI 53706, USA b Indiana University School of Medicine, Gary, IN, USA Received 23 April 2004; accepted in revised form 19 November 2004 Available online 4 January 2005
Abstract Nerve growth factor in the tear film and corneal epithelium is hypothesized to play an important role in ocular surface maintenance and corneal wound healing. The purpose of this study was to determine the expression of nerve growth factor and its high affinity (trkA) receptor in tears, cornea, and lacrimal glands of normal dogs, the modulation of nerve growth factor and its trkA receptor during corneal wound healing, and the effect of topical nerve growth factor application on canine corneal epithelial wound healing. In the first of three experiments, the nerve growth factor content of tears, corneal epithelium, lacrimal gland, and 3rd eyelid gland was determined in normal dogs by enzymelinked immunosorbent assay and the expression of nerve growth factor and its trkA receptor were evaluated in the cornea and lacrimal glands by immunohistochemistry. In a second experiment, unilateral corneal epithelial defects were created, and tissues were evaluated for changes in nerve growth factor or trkA expression for 1 week. In a third experiment, bilateral corneal epithelial defects were created and the right eyes in each animal were treated 4 times daily with either recombinant human nerve growth factor, murine nerve growth factor, or nerve growth factor-blocking antibody. The results of this study showed that nerve growth factor levels in normal dog tears, corneal epithelium, third eyelid gland and lacrimal gland were 15.4G4.6 ng mlK1, 33.5G12.3, 52.4G17.4 and 48.8G9.4 ng gK1, respectively. NGF and trkA receptors were identified by immunohistochemistry in all tissues examined. After unilateral corneal wounding, nerve growth factor concentration increased in the tears bilaterally for 3 days, especially in the wounded eye, and then returned to pre-wounding values. Nerve growth factor content, and immunohistochemical staining for nerve growth factor and trkA, increased significantly in the ipsilateral cornea epithelium following unilateral wounding. Nerve growth factor concentrations in lacrimal and third eyelid glands also increased bilaterally (p!0.01) after unilateral wounding. Time to wound closure and rate of epithelial migration did not differ significantly between nerve growth factor-treated, nerve growth factor antibody-treated, and control eyes. In conclusion, nerve growth factor is present under resting physiologic conditions in normal canine tears, and nerve growth factor and its trkA receptor are present under resting conditions in normal canine corneal epithelium, lacrimal gland and third eyelid gland. Nerve growth factor is elevated in the tears, cornea, and lacrimal glands after corneal epithelial wounding; however, topical application of nerve growth factor, or its blocking antibody does not modulate corneal wound healing in the normal dog eye. q 2004 Elsevier Ltd. All rights reserved. Keywords: neurotrophin; receptors; tears; lacrimal gland; corneal wound healing; canine
1. Introduction * Corresponding author. Dr Christopher J. Murphy, Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive W, Madison, WI 53706, USA. E-mail address: [email protected] (C.J. Murphy). 1 Present address: Department of Veterinary Medicine, College of Animal Resources Science, Kangwon National University, Chunchon, South Korea. 0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2004.11.013
The survival of primary sensory neurons depends on neurotrophins exerting their biological function by signaling through specific receptors of the trk family of tyrosine kinase receptors (Klein et al., 1991; Snider, 1994). Nerve growth factor (NGF) represents the first isolated and best characterized member of a growing family of neurotrophins
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that is best known for its profound effects on cells of the nervous system (Barde, 1990). In its active form, NGF isolated and purified from male mouse salivary gland is a dimer weighing approximately 26 kDa (Bocchini and Angeletti, 1969). The primary receptors for NGF are the high affinity tyrosine kinase A (trkA) and low affinity p75 receptors. In recent years, numerous studies have provided evidence that NGF also exerts effects on various non-neural cells (Matsuda et al., 1998; Villoslada et al., 2000; Dissen et al., 2001). NGF has been shown to play important roles in such diverse biological activities as modulation of cell proliferation (Descamps et al., 2001; Raychaudhuri et al., 2001; Tsuboi et al., 2001), migration (Micera et al., 2001; Krygier and Djakiew, 2002), and differentiation (Paul et al., 1996; Vawter et al., 1996; Nosrat et al., 1997; Murphy et al., 2000) in various cells. A multitude of trophic factors including NGF have also been identified in the corneas of humans and other vertebrates (Wilson et al., 1992, 1994; Campbell et al., 2000; Lambiase et al., 2000a,b; Murphy et al., 2000). In vivo studies have shown NGF and trkA receptors to be present in human, rabbit and rat cornea, and the presence of NGF in the tear film has also been reported in humans (Lambiase et al., 2000b; You et al., 2000). Additionally, it has been shown in vitro that primary corneal epithelial cells produce NGF (Lambiase et al., 2000b). In preliminary studies, we have also documented that NGF not only is synthesized by SV-40 human corneal epithelial cells (SV-40 HCEC) (Campbell et al., 2000), but also plays an important role in migration and proliferation of the cells (Murphy et al., 2000). In clinical studies, NGF has recently gained attention for the treatment of human patients with chronic corneal epithelial defects. Topical exogenous NGF has been reported to restore corneal integrity in human patients with immune or neurotrophic corneal ulcers (Lambiase et al., 1998; Bonini et al., 2000; Lambiase et al., 2000a). These observations suggest that tear and corneal epithelial NGF may play an important functional role in corneal wound healing. Why study dogs? Dogs are the most commonly seen patients in veterinary practice to develop spontaneous chronic corneal epithelial defects (SCCED). The clinical features and histopathological findings have been recently described and shown to have many similarities to humans (Bentley et al., 2001; Murphy et al., 2001). The features of SCCED are distinct from changes caused solely by the presence of a chronic corneal epithelial defect (Bentley et al., 2002). Recently, the efficacy of Substance P (SP)G insulin-like growth factor (IGF-1) in the treatment of humans with chronic corneal epithelial defects was evaluated in the dog SCCED model. (Murphy et al., 2001). The role of NGF on corneal wound healing in the dog, however, has not yet been studied. When studying homeostasis and wound healing in the cornea, it is important to consider potential interactions
between the ocular surface and the lacrimal tissues that produce the tears that bathe and protect the anterior surface of the eye. Therefore, identifying the change of NGF content in lacrimal tissue is important in improving our understanding of its exocrine function associated with corneal wound healing. To explore the role of corneal and tear NGF in corneal epithelial wound healing in the dog cornea, we determined basal expression of NGF in tears, cornea and lacrimal tissue and its modulation by epithelial wounding. Additionally, we evaluated effects of topical NGF and NGF blocking antibody on corneal epithelial wound healing in the dog.
2. Materials and methods All animals were treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use committee of the University of Wisconsin-Madison approved all protocols. 2.1. Effect of corneal wounding on NGF content Tears were collected from both eyes of 12 normal, control Beagle dogs twice a day for 2 days prior to wounding, using sterile, soft, fine diameter silicone tubing attached to a 3 ml syringe. Prior to wounding, all dogs were sedated with 1 mg kgK1 morphine and 0.05 mg kgK1 acepromazine intramuscularly (IM). Six mm diameter circular axial epithelial defects were created in the right eyes using mechanical debridement with an excimer laser spatula (nZ6) while the left eyes served as unwounded controls. Previous work in our laboratory has shown that debridement with an excimer laser spatula removes all of the epithelial cells but not the basement membrane (Woo, unpublished data). Debrided epithelium was collected and stored in 50 ml of PBS immediately after surgery and placed on dry ice. Morphine (0.5 mg kgK1, IM) was given for pain as needed for 3 days after surgery. No topical analgesics or antibiotics were given subsequent to wounding. Tears were collected once a day from both eyes for 1 week following debridement. After 1 week the dogs were euthanized with an IV injection of pentobarbital (100–200 mg kgK1) and then the corneas, lacrimal glands, and 3rd eyelid glands of each dog were rapidly collected and stored at K708C until processing for the measurement of NGF. Identical tissues were collected from six other (non-wounded) normal, control dogs euthanized for reasons other than this study. 2.2. Immunohistochemistry Corneas, third eyelid glands, and lacrimal glands were collected bilaterally from three control dogs and from three dogs that were subjected to unilateral corneal epithelial
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debridement 48 hr previously All tissues were immersionfixed in 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer at pH 7.4 for 30 min at room temperature. After fixation, 10 mm-thick sections were cut with a cryostat and processed for immunohistochemistry using primary antibodies raised against mouse NGF (1:800) or guinea pig trkA (1:400, both antisera from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Subsequent immunohistochemical visualization of the proteins was achieved by using a Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the substrate. Specificity of the immunohistochemical procedure was confirmed by incubating randomly selected tissue sections in the absence of the appropriate primary antibody. Specificity of NGF immunostaining was further demonstrated by processing several tissue sections in NGF primary antiserum that had been pre-absorbed with a five-fold higher concentration of NGF blocking peptide (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). 2.3. NGF immunoassay procedure Tissues were rapidly dissected and weighed, and tissue NGF was extracted according to the method of Zettler et al. (1996) with minor modifications. Briefly, the samples were homogenized in 0.5 ml ice cold extraction medium consisting of Tris–HCl (100 mM), NaCl (0.4 M), BSA (1%), sodium azide (0.05%), Triton X-100 (1%), phenylmethylsulphonyl fluoride (1 m M), EDTA (4 m M ) and aprotinin (0.09 TIU mlK1), pH 7.0. After homogenization, samples were spun at 3000 rpm for 15 min at 48C and supernatants were collected for NGF immunoassay. Tears were directly placed in the NGF ELISA. NGF concentrations were determined using a commercially available ELISA (NGF EmaxTM immunoassay system, Promega, Madison, WI), which has a detection limit of 5 pg mlK1, and expressed as ng mlK1 of tears or ng gK1 of tissue. All results were expressed as the meanGSD. Differences were assessed using a paired t-test. 2.4. Effect of NGF and anti-NGF blocking antibody on corneal epithelial wound healing Sixteen young adult female beagle dogs with normal ophthalmic examinations were used in the experiment. All dogs were sedated with 1 mg kgK1 IM morphine and 0.05 mg kgK1 IM acepromazine. Six millimetre axial corneal epithelial defects were created using mechanical debridement in both eyes of each dog. The animals were divided into three groups. The right eyes in each group were treated every 6 hr with either 200 mg mlK1 of recombinant human NGF (rh NGF, provided by Genentech, San Francisco, CA; nZ4), 200 mg mlK1 murine NGF (provided by Dr Rama, Hospital of Venice, Italy; nZ6), or 600 mg mlK1 anti-NGF blocking antibody (provided by Genentech, San Francisco, CA; nZ6) in PBS. The left eye
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of each animal was treated with an equimolar concentration of bovine serum albumin (BSA) in PBS to control for nonspecific protein effects. The size of the defect was measured by instillation of fluorescein and subsequent image capture and analysis using NIH Image software. Measurements were taken every 6 hr until the defects were healed. All images were taken at a fixed focal length under a cobalt blue filter. The healing rates were expressed as the linear decrease of the spherical wound radius per hour corrected for the corneal curvature of the canine eye (Woo et al., 2001). All results were expressed as the meanGSD. Differences were assessed using a paired t-test.
3. Results 3.1. NGF and trkA in control dogs NGF was detected by enzyme linked immunoassay in the tears, corneal epithelium, lacrimal gland and third eyelid gland of control dogs (Table 1). NGF and its trkA receptor were also demonstrated by immunohistochemistry in control dog corneas, lacrimal glands, and third eyelid glands. NGF and trkA-immunoreactive (-IR) cells were observed in all layers of the corneal epithelium; however, the highest levels of protein expression were found in the basal epithelial cells, especially in the limbus (Fig. 1a and b). In contrast, corneal keratocytes were devoid of NGF and trkA immunostaining, except for a few faintly labeled keratocytes in the limbal stroma. A modest number of NGF-IR nerves, and a small number of trkA-IR nerves, were seen in the corneal epithelium and in the anterior stroma (Figs. 1 and 2). The lacrimal and third eyelid glands contained a variety of NGF- and trkA-IR cells, including acinar cells, ductal cells, and occasional myoepithelial and vascular endothelial cells (Fig. 3). NGF- and trkA-IR cells in the lacrimal gland were distributed in a disparate manner. In any given secretory unit, some lacrimal acinar cells were densely stained, others were faintly stained, and still others were totally devoid of staining. The specificity of the immunohistochemical procedure used in this study was confirmed by the controls. Omission of the NGF or trkA primary antisera, or preabsorption of the NGF antisera with the NGF blocking peptide, eliminated all staining from the tissue sections.
Table 1 NGF concentrations measured in samples from normal dogs Sample Tears (ng ml ) Corneal epithelium (ng gK1) Third eyelid gl. (ng gK1) Lacrimal gl. (ng gK1) K1
Mean
SD
n
15.4 33.5 52.4 48.8
4.6 12.3 17.4 9.4
24 18 12 12
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Fig. 1. NGF-(a, b) and trkA-(c, d) immunoreactive cells in the corneal and limbal epithelium of a control dog. NGF and trkA are expressed most heavily in the basal epithelial cells especially in the limbus (arrows in b and d.)
3.2. Effect of corneal wounding on NGF and trkA expression Tears. NGF tear concentration increased significantly, and bilaterally, for 3 days after unilateral epithelial wounding (Fig. 4). For the first 48 hr, NGF tear concentration in the wounded eye was about 5-6 times higher than in the contralateral unwounded eye. By day 3, tear NGF content began to decline and by days 4–7 post-wounding, NGF tear concentrations had returned, bilaterally, to their normal pre-wounding levels. Cornea. Wounded corneas in the present investigation were completely resurfaced with a thin layer of new epithelium approximately 72 hr after epithelial debridement. Immunohistochemical analyses of corneas at 48 hr post-wounding revealed a substantial increase in NGF and trkA expression in all corneal epithelial cells bordering the residual wound margin and extending backwards approximately 1mm from the wound edge (Fig. 5a and c). Further from the wound margin, NGF and trkA were upregulated in virtually every cell of the basal epithelium, extending all the way to the limbus. Modest numbers of NGF- and trkA-IR keratocytes were observed in the subepithelial stroma immediately beneath the debrided area (Fig. 5a and c), and a profusion of immunoreactive keratocytes was seen in the posterior and peripheral stroma. In contrast, NGF and trkA expression in epithelial cells and keratocytes from contralateral, non-wounded corneas was qualitatively unchanged from normal corneas (Fig. 5b and d).
Radioimmunoassay confirmed and extended the results of the immunohistochemical analyses. One week after unilateral epithelial debridement, epithelial NGF content was significantly elevated on the wounded side relative to the contralateral non-wounded side and compared to normal corneas (Fig. 6). NGF concentrations in non-wounded and normal epithelium were not significantly different. Lacrimal and 3rd eyelid glands. Immunohistochemical analyses of the lacrimal and third-eyelid glands 48 hr after
Fig. 2. NGF-IR nerves (arrows) in the corneal epithelium and subepithelial stroma of a control dog.
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Fig. 3. NGF-(a) and trkA-(b) IR cells in the lacrimal gland of a control dog. The acinar cells are variably stained, such that some express substantial amounts of NGF or trkA (arrows), while others express lesser amounts of these proteins. Asterisk: lumen of a secretory unit (acinus).
cornea epithelial injury revealed no obvious qualitative or quantitative differences in NGF or trkA expression compared to control glands. In marked contrast, 1 week after unilateral corneal epithelial wounding radioimmunoassay revealed a significant, bilateral increase in lacrimal and 3rd eyelid gland NGF concentration compared to normal levels (Fig. 7). The increase in glandular NGF concentration did not differ significantly between the two sides (Fig. 7). 3.3. Effect of exogenous NGF and anti-NGF blocking antibody on corneal epithelial wound healing rate The mean wound healing rates for all groups of treated and control eyes were similar. Time to wound closure and rate of epithelial migration were not significantly different between rh NGF-treated, murine NGF-treated, antiNGF antibody-treated, and control (BSA-treated) eyes (Table 2).
immunohistochemical localization suggests a potentially important role for NGF in corneal epithelial cell cycling and, by extension, epithelial maintenance and homeostasis (Lambiase et al., 1998; Touhami et al., 2002). The identical patterns of epithelial NGF and trkA immunolocalization seen here in the dog cornea and in other mammalian corneas by other workers suggests that most, if not all, basal epithelial cells express both proteins. These observations support the hypothesis that NGF and trkA act in an autocrine or paracrine manner to provide trophic support to these cells (Lambiase et al., 2000b). In culture, exogenous NGF stimulates proliferation, migration, and differentiation of rabbit and SV-40 transformed human corneal epithelial cells (Kruse and Tseng, 1993; Murphy et al., 2000); however, its mitogenic effect is considerably weaker than that of other ocular growth factors such as epidermal growth factor (You et al., 2000). Studies in skin keratinocytes, which are in many ways analogous to corneal epithelial cells, suggest that NGF also prevents basal epithelial cell apoptosis (Pincelli et al., 1997).
4. Discussion 4.1. NGF and trkA expression in normal dogs The results of this study have demonstrated for the first time, by ELISA and immunohistochemistry, the presence of NGF and its high affinity trkA receptor in the normal dog corneal epithelium. The data confirm previous reports of NGF and trkA expression in rat, rabbit, and human corneal epithelium (Lambiase et al., 2000b, 2002; Touhami et al., 2002; Campbell et al., 2000). In the dog cornea, NGF- and trkA are expressed in the highest concentrations in the basal epithelial cells, especially near and in the limbus where the corneal stem cell population is located. This pattern of
Fig. 4. Tear NGF content after unilateral corneal epithelial debridement. Note increase in NGF after unilateral wounding. *p!0.001 comparing the value to baseline; **p!0.0001 comparing the value to baseline; !p!0.001 comparing the value to the unwounded.
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Fig. 5. NGF and trkA expression in the wounded (a, c, e) and contralateral non-wounded (b, d) cornea, 48 hr after unilateral epithelial debridement. Note the substantial increase in NGF and trkA expression in the reparative corneal epithelium (a, c) and in keratocytes (arrows in a, c, e) on the wounded side. The thinner epithelium is consistent with epithelial migration without extensive proliferation 48 hr post wounding.
The extreme paucity of NGF and trkA immunostaining in normal dog keratocytes in the present study is at odds with prior reports in other species, including, human, rat, and rabbit corneas (Lambiase et al., 2000b; Lambiase et al., 2002). It is possible that under resting physiological conditions dog keratocytes contain levels of NGF
and trkA that are below the limits of immunodetection. The presence in the same tissue sections of positively stained corneal epithelial cells and stromal nerves argues against a deficiency of the immunohistochemical method. The NGF- and trkA-IR nerve fibers seen here in the dog cornea are morphologically indistinguishable from
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Fig. 6. Corneal epithelium NGF content 1 week after corneal epithelial debridement. Note the increase of NGF in the epithelium of wounded corneas. *pZ0.024 comparing the value to baseline; !pZ0.029 comparing the value to the unwounded contralateral eyes.
Fig. 7. Lacrimal tissue NGF content 1 week after corneal epithelial debridement. Note that NGF concentrations are higher than unwounded controls both ipsilateral and contralateral to the wounded side. *p!0.001 comparing the value to normal values; **p!0.0001 comparing the value to normal values.
the canine corneal nerve fibers described in our earlier study (Marfurt et al., 2001). The source of the NGF contained within these nerves is a matter of speculation. Corneal nerves supply a rich innervation to the corneal epithelium and perhaps NGF released by the epithelial cells is internalized by the nerve terminals and transported retrogradely into the axons. Alternatively, trigeminal ganglion neurons are able to produce endogenous NGF and trkA and can transport these proteins into their peripheral axons (Jacobs and Miller, 1999). The simultaneous demonstration within the cornea of NGF- and trkA immunoreactive nerves
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and epithelial cells suggests intriguing possibilities for mutual trophic interactions. For example, epitheliumderived NGF may promote peripheral nerve maintenance, regenerative sprouting, and expression of trophic neuropeptides such as substance P and CGRP (MacLean et al., 1988; Donnerer et al., 1992), while NGF released from corneal nerves may promote epithelial homeostasis and reparative processes associated with wound healing. The results of the present study have demonstrated, by both ELISA and immunohistochemistry, the presence of NGF in normal dog lacrimal and third eyelid glands and corroborates an earlier report of NGF mRNA in the human lacrimal gland (Nguyen et al., 1997). In fact, levels of NGF were highest in lacrimal glands and glands of the third eyelid. Combined with the presence of NGF in the tear film, this data suggests that lacrimal tissue is an important source of NGF in vivo. Analogous patterns of NGF and trkA immunostaining suggests that individual acinar cells probably express both proteins and that lacrimal NGF likely acts in an autocrine or paracrine fashion to modulate gland activity. The immunohistochemical data presented here reveals that individual acinar cells in the same secretory unit exhibit substantial cell-to-cell differences in resting levels of NGF and trkA expression and/or storage. Because acinar cell secretion is under the control of the parasympathetic nervous system, the cell-to-cell differences in acinar protein expression seen here may reflect distributional or functional differences in parasympathetic innervation (Williams et al., 1994). Finally, the current study has shown that NGF is present in normal canine tears, confirming similar observations from tear fluids in human subjects (Vesaluoma et al., 2000). The results of the ELISA and immunohistochemical studies strongly suggest that tear fluid NGF is contributed from multiple sources, including, lacrimal and third eyelid glands, corneal epithelium and perhaps corneal and conjunctival nerves. The demonstration of NGF in canine tear fluid under resting physiologic conditions suggests that NGF, at appropriate concentrations and perhaps in synergism with other growth factors in the tear film, plays an important role in corneal epithelial maintenance and homeostasis.
Table 2 MeanGSD epithelial healing rates and time to healing after corneal epithelial debridement Treatments
Murine NGF (nZ6) Rh NGFc (nZ4) Anti-NGF Ab. (nZ6) a b c
Healing ratea (mm hrK1)
Time to heal (hr)
Treated
Controlb
p value
Treated
Controlb
p value
67.9G13.1 62.3G17.9 62.8G16.8
61.2G12.8 64.8G13.5 64.6G12.4
0.40 0.46 0.64
53.8G7.2 60.6G4.6 64.7G8.9
59.5G14.5 56.2G8.3 56.8G7.4
0.40 0.18 0.12
The rates were calculated by the decrease in wound radius. Treated with bovine serum albumin. Recombinant human Nerve Growth Factor.
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4.2. Alterations in corneal NGF and trkA expression following epithelial wounding Unilateral corneal epithelial debridement produced a significant ipsilateral increase in canine epithelial NGF expression that was demonstrated in the present study by two independent methods, i.e. immunohistochemistry (2 days post-wounding) and ELISA (7 days post-wounding). These findings confirm previous reports of increased epithelial NGF expression and/or binding in rats after corneal epithelial debridement (Lambiase et al., 2000b) and after skin wounding (Constantinou et al., 1994; Matsuda et al., 1998). The simultaneous increases in NGF and trkA expression seen here in the dog cornea after wounding occur preferentially in corneal epithelial cells near the wound margin and throughout the basal epithelium. This pattern of NGF/trkA upregulation is notable because it occurs primarily in cells that are actively engaged in reparative behaviors such as migration and proliferation. It is tempting to speculate that by augmenting activity in the endogenous NGF/trkA autocrine-paracrine system, migratory and proliferating corneal epithelial cells can further increase their responsivity to the wound-promoting effects of NGF. In the present study, epithelial scrape injury also produced, as revealed by immunohistochemical analysis 48 hr later, a widespread and pronounced upregulation of NGF and trkA expression in stromal keratocytes. One of the first responses to corneal epithelial debridement is the loss of keratocytes from the underlying stroma (Wilson et al., 1996; Zhao et al., 2001) and in the current study the presence of a largely ‘cell-free’ zone beneath the epithelial injury site is confirmed, although small numbers of scattered keratocytes persist. Whether the latter cells are resident keratocytes that fail to degenerate, or keratocytes that have migrated into the area in the past 48 hr, is unknown. Regardless, these keratocytes, and virtually every keratocyte in the corneal stroma outside the cell-poor zone, stain prominently for NGF and/or trkA. The sheer numbers of keratocytes that express either NGF or trkA in serial tissue sections strongly suggests that most, if not all, keratocytes express both proteins after wounding. It is tempting to speculate that keratocytes may utilize this NGF/trkA autocrine-paracrine system to promote migration and proliferation and thereby facilitate repopulation of the cell-poor zone. Future work should examine alterations in keratocye apoptosis by NGF following epithelial injury. 4.3. NGF alterations in the lacrimal gland and tears Following corneal epithelial scrape injuries, the lacrimal gland increases its production of several growth factors, including, EGF, HGF, TGF-b and KGF (Wilson et al., 1999; Lim et al., 2003). The results of the present ELISA study adds to this list by showing that NGF production is also increased, not only in the lacrimal gland but also in the third eyelid gland. The mechanism by which corneal epithelial
injury stimulates lacrimal gland NGF production is uncertain, but most likely involves a reflex neural circuit comprised of corneal sensory nerves, brainstem connections, and lacrimal parasympathetic (cholinergic) innervation. Cholinergic stimulation also increases EGF secretion by the lacrimal gland (Wilson et al., 1991). Unilateral wounding of the dog cornea also causes NGF concentrations to increase in the contralateral lacrimal gland, third eyelid gland, and tear fluid. The contralateral response may also be mediated neuronally (through crossed brainstem connections), or perhaps systemically. In support of the latter hypothesis, it has been shown that skin wounding in mice causes the salivary glands to release NGF into the systemic circulation and increases serum NGF levels (Matsuda et al., 1998). The rapid and significant increases in lacrimal gland and tear NGF levels seen here after corneal epithelial wounding provide further support for the concept of the ocular surface as a ‘functional unit.’ According to this model, corneal epithelial maintenance and wound healing depend on the coordinated biological activities of the cornea, tear film, main and accessory lacrimal glands, and their interconnecting reflex neural circuits (Stern et al., 1998; Mathers, 2000). Furthermore, the abrupt decrease of NGF at 48 hr, when the wounds were almost healed, may indicate a downregulation once the epithelium is reformed. 4.4. Topical NGF and corneal wound healing Neither topical NGF nor topical anti-NGF blocking antibody altered the corneal epithelial wound healing rate when applied to otherwise healthy dog eyes in this study. These findings contrast with previous reports from another laboratory. Lambiase and coworkers reported that topical application of murine NGF accelerated corneal epithelial wound healing, and anti-NGF antibody attenuated wound healing, in the rabbit cornea (Lambiase et al., 2000b). In clinical studies by these same authors, topical NGF was reported to accelerate the healing of corneal ulcers in patients with neurotrophic keratitis from various causes (Lambiase et al., 1998; Bonini et al., 2000). The reasons for the apparent discrepancy between our data and those of Lambiase et al. are not clear, but several factors may contribute to the differences. It is possible that endogenous NGF concentrations at the wound site in normal, control dogs after corneal wounding may already be optimal and thus supplementation through topical application would not improve the wound healing response. In contrast, the positive responses to topically applied NGF observed in human corneas occurred in patients with preexisting epithelial defects and ocular surface environments that may have been deficient in one or more essential growth factors (Lambiase et al., 1998; Bonini et al., 2000). Another possible contributing factor is the relative specificity of the agents used. It is possible that the murine 2.5S NGF, rh NGF and blocking antibody used in this study do
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not bind with the same avidity to canine receptors, or that intracellular pathways activated subsequent to binding are different. NGF with different forms has been isolated from various sources and methods including 2.5S NGF with 26 kDa, and 7S NGF with 14 or 116 kDa from mouse salivary, submaxillary, or submandibular gland, respectively (Young et al., 1978; Shooter, 2001). The murine NGF used in this study has been shown to act on other species such as mice, rats, rabbits and humans (Li et al., 1980; Kruse and Tseng, 1993; Lambiase et al., 2000b); however, species variability in response to NGF from different sources have also been reported. Exogenous 7S NGF and HMW-NGF obtained from mouse salivary gland affected skin wound healing differently when tested in the Syrian hamster and mouse (Li et al., 1980). Collectively, these data suggest that the wound healing properties of topical NGF may exhibit significant species-specific differences. The results of the current study have shown that NGF is present in measurable quantities in normal dog tears, corneal epithelium, lacrimal gland, and third eyelid gland. Furthermore, NGF concentrations in these tissues are significantly increased, either ipsilaterally or bilaterally, following unilateral corneal epithelial wounding. Application of exogenous NGF, however, does not alter wound healing in vivo in the dog.
Acknowledgements This work was supported by a grant from the National Eye Institute (EY 10841-01) and was presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology in Fort Lauderdale, Florida, May 2001.
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defects (SCCED) in dogs: clinical features, innervation, and effect of topical SP, with or without IGF-1. Invest. Ophthalmol. Vis. Sci. 42, 2252–2261. Nguyen, D.H., Beuerman, R.W., Thompson, H.W., DiLoreto, D.A., 1997. Growth factor and neurotrophic factor mRNA in human lacrimal gland. Cornea 16, 192–199. Nosrat, C.A., Fried, K., Lindskog, S., Olson, L., 1997. Cellular expression of neurotrophin mRNAs during tooth development. Cell Tissue Res. 290, 569–580. Paul, A.B., Grant, E.S., Habib, F.K., 1996. The expression and localisation of beta-nerve growth factor (beta-NGF) in benign and malignant human prostate tissue: relationship to neuroendocrine differentiation. Br. J. Cancer 74, 1990–1996. Pincelli, C., Yaar, M., 1997. Nerve growth factor: its significance in cutaneous biology. J. Invest. Derm. Symp. Proc. 2, 31–36. Raychaudhuri, S.K., Raychaudhuri, S.P., Weltman, H., Farber, E.M., 2001. Effect of nerve growth factor on endothelial cell biology: proliferation and adherence molecule expression on human dermal microvascular endothelial cells. Arch. Dermatol. Res. 293, 291–295. Shooter, E.M., 2001. Early days of the nerve growth factor proteins. Annu. Rev. Neurosci. 24, 601–629. Snider, W.D., 1994. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell 77, 627–638. Stern, M.E., Beuerman, R.W., Fox, R.I., Gao, J., Mircheff, A.K., Pflugfelder, S.C., 1998. A unified theory of the role of the ocular surface in dry eye. Adv. Exp. Med. Biol. 438, 643–651. Touhami, A., Grueterich, M., Tseng, S.C., 2002. The role of NGF signaling in human limbal epithelium expanded by amniotic membrane culture. Invest. Ophthalmol. Vis. Sci. 43, 987–994. Tsuboi, Y., Nakanishi, T., Takano-Yamamoto, T., Miyamoto, M., Yamashiro, T., Takigawa, M., 2001. Mitogenic effects of neutrophins on a periodontal ligament cell line. J. Dent. Res. 80, 881–886. Vawter, M.P., Basaric-Keys, J., Li, Y., Lester, D.S., Lebovics, R.S., Lesch, K.P., Kulaga, H., Freed, W.J., Sunderland, T., Wolozin, B., 1996. Human olfactory neuroepithelial cells: tyrosine phosphorylation and process extension are increased by the combination of IL-1beta, IL6, NGF, and bFGF. Exp. Neurol. 142, 179–194. Vesaluoma, M., Muller, L., Gallar, J., Lambiase, A., Moilanen, J., Hack, T., Belmonte, C., Tervo, T., 2000. Effects of oleoresin capsicum pepper spray on human corneal morphology and sensitivity. Invest. Ophthalmol. Vis. Sci. 41, 2138–2147. Villoslada, P., Hauser, S.L., Bartke, I., Unger, J., Heald, N., Rosenberg, D., Cheung, S.W., Mobley, W.C., Fisher, S., Genain, C.P., 2000. Human
nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system. J. Exp. Med. 191, 1799–1806. Williams, R.M., Singh, J., Sharkey, K.A., 1994. Innervation and mast cells of the rat lacrimal gland: the effects of age. Adv Exp Med Biol. 350, 67–74. Wilson, S.E., Lloyd, S.A., Kennedy, R.H., 1991. Basic fibroblast growth factor (FGFb) and epidermal growth factor (EGF) receptor messenger RNA production in human lacrimal gland. Invest. Ophthal. Vis. Sci. 32, 2816–2820. Wilson, S.E., He, Y.G., Lloyd, S.A., 1992. EGF, EGF receptor, basic FGF, TGF beta-1, and IL-1 alpha mRNA in human corneal epithelial cells and stromal fibroblasts. Invest. Ophthalmol. Vis. Sci. 33, 1756–1765. Wilson, S.E., Schultz, G.S., Chegini, N., Weng, J., He, Y.G., 1994. Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp. Eye. Res. 59, 63–71. Wilson, S.E., He, Y-G., Weng, J., Li, Q., McDowall, A.W., Vital, M., Chwang, E.L., 1996. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization. Exp. Eye Res. 62, 325–338. Wilson, S.E., Liang, Q., Kim, W.J., 1999. Lacrimal gland HGF, KGF, and EGF mRNA levels increase after corneal epithelial wounding. Invest. Ophthal. Vis. Sci. 40, 2185–2190. Woo, H.M., Kim, M.S., Kweon, O.K., Kim, D.Y., Nam, T.C., Kim, J.H., 2001. Effects of amniotic membrane on epithelial wound healing and stromal remodelling after excimer laser keratectomy in rabbit cornea. Br. J. Ophthalmol. 85, 345–349. You, L., Kruse, F.E., Volcker, H.E., 2000. Neurotrophic factors in the human cornea. Invest. Ophthalmol. Vis. Sci. 41, 692–702. Young, M., Saide, J.D., Murphy, R.A., 1978. Blanchard, Nerve growth factor: multiple dissociation products in homogenates of the mouse submandibular gland. Purification and molecular properties of the intact undissociated form of the protein. Biochemistry 17, 1490–1498. Zettler, C., Bridges, D.C., Zhou, X.F., Rush, R.A., 1996. Detection of increased tissue concentrations of nerve growth factor with an improved extraction procedure. J. Neurosci. Res. 46, 581–594. Zhao, J., Nagasaki, T., Maurice, D.M., 2001. Role of tears in keratocyte loss after epithelial removal in mouse cornea. Invest. Ophthalmol. Vis. Sci. 42, 1743–1749.
Nerve growth factor and corneal wound healing in dogs Heung-Myong Wooa,1, Ellison Bentleya, Sean F. Campbella, Carl F. Marfurtb, Christopher J. Murphya,* a
Department of Surgical Sciences, School of Veterinary Medicine, School of Medicine, University of Wisconsin-Madison, 2015 Linden Drive W, Madison, WI 53706, USA b Indiana University School of Medicine, Gary, IN, USA Received 23 April 2004; accepted in revised form 19 November 2004 Available online 4 January 2005
Abstract Nerve growth factor in the tear film and corneal epithelium is hypothesized to play an important role in ocular surface maintenance and corneal wound healing. The purpose of this study was to determine the expression of nerve growth factor and its high affinity (trkA) receptor in tears, cornea, and lacrimal glands of normal dogs, the modulation of nerve growth factor and its trkA receptor during corneal wound healing, and the effect of topical nerve growth factor application on canine corneal epithelial wound healing. In the first of three experiments, the nerve growth factor content of tears, corneal epithelium, lacrimal gland, and 3rd eyelid gland was determined in normal dogs by enzymelinked immunosorbent assay and the expression of nerve growth factor and its trkA receptor were evaluated in the cornea and lacrimal glands by immunohistochemistry. In a second experiment, unilateral corneal epithelial defects were created, and tissues were evaluated for changes in nerve growth factor or trkA expression for 1 week. In a third experiment, bilateral corneal epithelial defects were created and the right eyes in each animal were treated 4 times daily with either recombinant human nerve growth factor, murine nerve growth factor, or nerve growth factor-blocking antibody. The results of this study showed that nerve growth factor levels in normal dog tears, corneal epithelium, third eyelid gland and lacrimal gland were 15.4G4.6 ng mlK1, 33.5G12.3, 52.4G17.4 and 48.8G9.4 ng gK1, respectively. NGF and trkA receptors were identified by immunohistochemistry in all tissues examined. After unilateral corneal wounding, nerve growth factor concentration increased in the tears bilaterally for 3 days, especially in the wounded eye, and then returned to pre-wounding values. Nerve growth factor content, and immunohistochemical staining for nerve growth factor and trkA, increased significantly in the ipsilateral cornea epithelium following unilateral wounding. Nerve growth factor concentrations in lacrimal and third eyelid glands also increased bilaterally (p!0.01) after unilateral wounding. Time to wound closure and rate of epithelial migration did not differ significantly between nerve growth factor-treated, nerve growth factor antibody-treated, and control eyes. In conclusion, nerve growth factor is present under resting physiologic conditions in normal canine tears, and nerve growth factor and its trkA receptor are present under resting conditions in normal canine corneal epithelium, lacrimal gland and third eyelid gland. Nerve growth factor is elevated in the tears, cornea, and lacrimal glands after corneal epithelial wounding; however, topical application of nerve growth factor, or its blocking antibody does not modulate corneal wound healing in the normal dog eye. q 2004 Elsevier Ltd. All rights reserved. Keywords: neurotrophin; receptors; tears; lacrimal gland; corneal wound healing; canine
1. Introduction * Corresponding author. Dr Christopher J. Murphy, Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive W, Madison, WI 53706, USA. E-mail address: [email protected] (C.J. Murphy). 1 Present address: Department of Veterinary Medicine, College of Animal Resources Science, Kangwon National University, Chunchon, South Korea. 0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2004.11.013
The survival of primary sensory neurons depends on neurotrophins exerting their biological function by signaling through specific receptors of the trk family of tyrosine kinase receptors (Klein et al., 1991; Snider, 1994). Nerve growth factor (NGF) represents the first isolated and best characterized member of a growing family of neurotrophins
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that is best known for its profound effects on cells of the nervous system (Barde, 1990). In its active form, NGF isolated and purified from male mouse salivary gland is a dimer weighing approximately 26 kDa (Bocchini and Angeletti, 1969). The primary receptors for NGF are the high affinity tyrosine kinase A (trkA) and low affinity p75 receptors. In recent years, numerous studies have provided evidence that NGF also exerts effects on various non-neural cells (Matsuda et al., 1998; Villoslada et al., 2000; Dissen et al., 2001). NGF has been shown to play important roles in such diverse biological activities as modulation of cell proliferation (Descamps et al., 2001; Raychaudhuri et al., 2001; Tsuboi et al., 2001), migration (Micera et al., 2001; Krygier and Djakiew, 2002), and differentiation (Paul et al., 1996; Vawter et al., 1996; Nosrat et al., 1997; Murphy et al., 2000) in various cells. A multitude of trophic factors including NGF have also been identified in the corneas of humans and other vertebrates (Wilson et al., 1992, 1994; Campbell et al., 2000; Lambiase et al., 2000a,b; Murphy et al., 2000). In vivo studies have shown NGF and trkA receptors to be present in human, rabbit and rat cornea, and the presence of NGF in the tear film has also been reported in humans (Lambiase et al., 2000b; You et al., 2000). Additionally, it has been shown in vitro that primary corneal epithelial cells produce NGF (Lambiase et al., 2000b). In preliminary studies, we have also documented that NGF not only is synthesized by SV-40 human corneal epithelial cells (SV-40 HCEC) (Campbell et al., 2000), but also plays an important role in migration and proliferation of the cells (Murphy et al., 2000). In clinical studies, NGF has recently gained attention for the treatment of human patients with chronic corneal epithelial defects. Topical exogenous NGF has been reported to restore corneal integrity in human patients with immune or neurotrophic corneal ulcers (Lambiase et al., 1998; Bonini et al., 2000; Lambiase et al., 2000a). These observations suggest that tear and corneal epithelial NGF may play an important functional role in corneal wound healing. Why study dogs? Dogs are the most commonly seen patients in veterinary practice to develop spontaneous chronic corneal epithelial defects (SCCED). The clinical features and histopathological findings have been recently described and shown to have many similarities to humans (Bentley et al., 2001; Murphy et al., 2001). The features of SCCED are distinct from changes caused solely by the presence of a chronic corneal epithelial defect (Bentley et al., 2002). Recently, the efficacy of Substance P (SP)G insulin-like growth factor (IGF-1) in the treatment of humans with chronic corneal epithelial defects was evaluated in the dog SCCED model. (Murphy et al., 2001). The role of NGF on corneal wound healing in the dog, however, has not yet been studied. When studying homeostasis and wound healing in the cornea, it is important to consider potential interactions
between the ocular surface and the lacrimal tissues that produce the tears that bathe and protect the anterior surface of the eye. Therefore, identifying the change of NGF content in lacrimal tissue is important in improving our understanding of its exocrine function associated with corneal wound healing. To explore the role of corneal and tear NGF in corneal epithelial wound healing in the dog cornea, we determined basal expression of NGF in tears, cornea and lacrimal tissue and its modulation by epithelial wounding. Additionally, we evaluated effects of topical NGF and NGF blocking antibody on corneal epithelial wound healing in the dog.
2. Materials and methods All animals were treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use committee of the University of Wisconsin-Madison approved all protocols. 2.1. Effect of corneal wounding on NGF content Tears were collected from both eyes of 12 normal, control Beagle dogs twice a day for 2 days prior to wounding, using sterile, soft, fine diameter silicone tubing attached to a 3 ml syringe. Prior to wounding, all dogs were sedated with 1 mg kgK1 morphine and 0.05 mg kgK1 acepromazine intramuscularly (IM). Six mm diameter circular axial epithelial defects were created in the right eyes using mechanical debridement with an excimer laser spatula (nZ6) while the left eyes served as unwounded controls. Previous work in our laboratory has shown that debridement with an excimer laser spatula removes all of the epithelial cells but not the basement membrane (Woo, unpublished data). Debrided epithelium was collected and stored in 50 ml of PBS immediately after surgery and placed on dry ice. Morphine (0.5 mg kgK1, IM) was given for pain as needed for 3 days after surgery. No topical analgesics or antibiotics were given subsequent to wounding. Tears were collected once a day from both eyes for 1 week following debridement. After 1 week the dogs were euthanized with an IV injection of pentobarbital (100–200 mg kgK1) and then the corneas, lacrimal glands, and 3rd eyelid glands of each dog were rapidly collected and stored at K708C until processing for the measurement of NGF. Identical tissues were collected from six other (non-wounded) normal, control dogs euthanized for reasons other than this study. 2.2. Immunohistochemistry Corneas, third eyelid glands, and lacrimal glands were collected bilaterally from three control dogs and from three dogs that were subjected to unilateral corneal epithelial
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debridement 48 hr previously All tissues were immersionfixed in 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer at pH 7.4 for 30 min at room temperature. After fixation, 10 mm-thick sections were cut with a cryostat and processed for immunohistochemistry using primary antibodies raised against mouse NGF (1:800) or guinea pig trkA (1:400, both antisera from Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Subsequent immunohistochemical visualization of the proteins was achieved by using a Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the substrate. Specificity of the immunohistochemical procedure was confirmed by incubating randomly selected tissue sections in the absence of the appropriate primary antibody. Specificity of NGF immunostaining was further demonstrated by processing several tissue sections in NGF primary antiserum that had been pre-absorbed with a five-fold higher concentration of NGF blocking peptide (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). 2.3. NGF immunoassay procedure Tissues were rapidly dissected and weighed, and tissue NGF was extracted according to the method of Zettler et al. (1996) with minor modifications. Briefly, the samples were homogenized in 0.5 ml ice cold extraction medium consisting of Tris–HCl (100 mM), NaCl (0.4 M), BSA (1%), sodium azide (0.05%), Triton X-100 (1%), phenylmethylsulphonyl fluoride (1 m M), EDTA (4 m M ) and aprotinin (0.09 TIU mlK1), pH 7.0. After homogenization, samples were spun at 3000 rpm for 15 min at 48C and supernatants were collected for NGF immunoassay. Tears were directly placed in the NGF ELISA. NGF concentrations were determined using a commercially available ELISA (NGF EmaxTM immunoassay system, Promega, Madison, WI), which has a detection limit of 5 pg mlK1, and expressed as ng mlK1 of tears or ng gK1 of tissue. All results were expressed as the meanGSD. Differences were assessed using a paired t-test. 2.4. Effect of NGF and anti-NGF blocking antibody on corneal epithelial wound healing Sixteen young adult female beagle dogs with normal ophthalmic examinations were used in the experiment. All dogs were sedated with 1 mg kgK1 IM morphine and 0.05 mg kgK1 IM acepromazine. Six millimetre axial corneal epithelial defects were created using mechanical debridement in both eyes of each dog. The animals were divided into three groups. The right eyes in each group were treated every 6 hr with either 200 mg mlK1 of recombinant human NGF (rh NGF, provided by Genentech, San Francisco, CA; nZ4), 200 mg mlK1 murine NGF (provided by Dr Rama, Hospital of Venice, Italy; nZ6), or 600 mg mlK1 anti-NGF blocking antibody (provided by Genentech, San Francisco, CA; nZ6) in PBS. The left eye
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of each animal was treated with an equimolar concentration of bovine serum albumin (BSA) in PBS to control for nonspecific protein effects. The size of the defect was measured by instillation of fluorescein and subsequent image capture and analysis using NIH Image software. Measurements were taken every 6 hr until the defects were healed. All images were taken at a fixed focal length under a cobalt blue filter. The healing rates were expressed as the linear decrease of the spherical wound radius per hour corrected for the corneal curvature of the canine eye (Woo et al., 2001). All results were expressed as the meanGSD. Differences were assessed using a paired t-test.
3. Results 3.1. NGF and trkA in control dogs NGF was detected by enzyme linked immunoassay in the tears, corneal epithelium, lacrimal gland and third eyelid gland of control dogs (Table 1). NGF and its trkA receptor were also demonstrated by immunohistochemistry in control dog corneas, lacrimal glands, and third eyelid glands. NGF and trkA-immunoreactive (-IR) cells were observed in all layers of the corneal epithelium; however, the highest levels of protein expression were found in the basal epithelial cells, especially in the limbus (Fig. 1a and b). In contrast, corneal keratocytes were devoid of NGF and trkA immunostaining, except for a few faintly labeled keratocytes in the limbal stroma. A modest number of NGF-IR nerves, and a small number of trkA-IR nerves, were seen in the corneal epithelium and in the anterior stroma (Figs. 1 and 2). The lacrimal and third eyelid glands contained a variety of NGF- and trkA-IR cells, including acinar cells, ductal cells, and occasional myoepithelial and vascular endothelial cells (Fig. 3). NGF- and trkA-IR cells in the lacrimal gland were distributed in a disparate manner. In any given secretory unit, some lacrimal acinar cells were densely stained, others were faintly stained, and still others were totally devoid of staining. The specificity of the immunohistochemical procedure used in this study was confirmed by the controls. Omission of the NGF or trkA primary antisera, or preabsorption of the NGF antisera with the NGF blocking peptide, eliminated all staining from the tissue sections.
Table 1 NGF concentrations measured in samples from normal dogs Sample Tears (ng ml ) Corneal epithelium (ng gK1) Third eyelid gl. (ng gK1) Lacrimal gl. (ng gK1) K1
Mean
SD
n
15.4 33.5 52.4 48.8
4.6 12.3 17.4 9.4
24 18 12 12
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Fig. 1. NGF-(a, b) and trkA-(c, d) immunoreactive cells in the corneal and limbal epithelium of a control dog. NGF and trkA are expressed most heavily in the basal epithelial cells especially in the limbus (arrows in b and d.)
3.2. Effect of corneal wounding on NGF and trkA expression Tears. NGF tear concentration increased significantly, and bilaterally, for 3 days after unilateral epithelial wounding (Fig. 4). For the first 48 hr, NGF tear concentration in the wounded eye was about 5-6 times higher than in the contralateral unwounded eye. By day 3, tear NGF content began to decline and by days 4–7 post-wounding, NGF tear concentrations had returned, bilaterally, to their normal pre-wounding levels. Cornea. Wounded corneas in the present investigation were completely resurfaced with a thin layer of new epithelium approximately 72 hr after epithelial debridement. Immunohistochemical analyses of corneas at 48 hr post-wounding revealed a substantial increase in NGF and trkA expression in all corneal epithelial cells bordering the residual wound margin and extending backwards approximately 1mm from the wound edge (Fig. 5a and c). Further from the wound margin, NGF and trkA were upregulated in virtually every cell of the basal epithelium, extending all the way to the limbus. Modest numbers of NGF- and trkA-IR keratocytes were observed in the subepithelial stroma immediately beneath the debrided area (Fig. 5a and c), and a profusion of immunoreactive keratocytes was seen in the posterior and peripheral stroma. In contrast, NGF and trkA expression in epithelial cells and keratocytes from contralateral, non-wounded corneas was qualitatively unchanged from normal corneas (Fig. 5b and d).
Radioimmunoassay confirmed and extended the results of the immunohistochemical analyses. One week after unilateral epithelial debridement, epithelial NGF content was significantly elevated on the wounded side relative to the contralateral non-wounded side and compared to normal corneas (Fig. 6). NGF concentrations in non-wounded and normal epithelium were not significantly different. Lacrimal and 3rd eyelid glands. Immunohistochemical analyses of the lacrimal and third-eyelid glands 48 hr after
Fig. 2. NGF-IR nerves (arrows) in the corneal epithelium and subepithelial stroma of a control dog.
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Fig. 3. NGF-(a) and trkA-(b) IR cells in the lacrimal gland of a control dog. The acinar cells are variably stained, such that some express substantial amounts of NGF or trkA (arrows), while others express lesser amounts of these proteins. Asterisk: lumen of a secretory unit (acinus).
cornea epithelial injury revealed no obvious qualitative or quantitative differences in NGF or trkA expression compared to control glands. In marked contrast, 1 week after unilateral corneal epithelial wounding radioimmunoassay revealed a significant, bilateral increase in lacrimal and 3rd eyelid gland NGF concentration compared to normal levels (Fig. 7). The increase in glandular NGF concentration did not differ significantly between the two sides (Fig. 7). 3.3. Effect of exogenous NGF and anti-NGF blocking antibody on corneal epithelial wound healing rate The mean wound healing rates for all groups of treated and control eyes were similar. Time to wound closure and rate of epithelial migration were not significantly different between rh NGF-treated, murine NGF-treated, antiNGF antibody-treated, and control (BSA-treated) eyes (Table 2).
immunohistochemical localization suggests a potentially important role for NGF in corneal epithelial cell cycling and, by extension, epithelial maintenance and homeostasis (Lambiase et al., 1998; Touhami et al., 2002). The identical patterns of epithelial NGF and trkA immunolocalization seen here in the dog cornea and in other mammalian corneas by other workers suggests that most, if not all, basal epithelial cells express both proteins. These observations support the hypothesis that NGF and trkA act in an autocrine or paracrine manner to provide trophic support to these cells (Lambiase et al., 2000b). In culture, exogenous NGF stimulates proliferation, migration, and differentiation of rabbit and SV-40 transformed human corneal epithelial cells (Kruse and Tseng, 1993; Murphy et al., 2000); however, its mitogenic effect is considerably weaker than that of other ocular growth factors such as epidermal growth factor (You et al., 2000). Studies in skin keratinocytes, which are in many ways analogous to corneal epithelial cells, suggest that NGF also prevents basal epithelial cell apoptosis (Pincelli et al., 1997).
4. Discussion 4.1. NGF and trkA expression in normal dogs The results of this study have demonstrated for the first time, by ELISA and immunohistochemistry, the presence of NGF and its high affinity trkA receptor in the normal dog corneal epithelium. The data confirm previous reports of NGF and trkA expression in rat, rabbit, and human corneal epithelium (Lambiase et al., 2000b, 2002; Touhami et al., 2002; Campbell et al., 2000). In the dog cornea, NGF- and trkA are expressed in the highest concentrations in the basal epithelial cells, especially near and in the limbus where the corneal stem cell population is located. This pattern of
Fig. 4. Tear NGF content after unilateral corneal epithelial debridement. Note increase in NGF after unilateral wounding. *p!0.001 comparing the value to baseline; **p!0.0001 comparing the value to baseline; !p!0.001 comparing the value to the unwounded.
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Fig. 5. NGF and trkA expression in the wounded (a, c, e) and contralateral non-wounded (b, d) cornea, 48 hr after unilateral epithelial debridement. Note the substantial increase in NGF and trkA expression in the reparative corneal epithelium (a, c) and in keratocytes (arrows in a, c, e) on the wounded side. The thinner epithelium is consistent with epithelial migration without extensive proliferation 48 hr post wounding.
The extreme paucity of NGF and trkA immunostaining in normal dog keratocytes in the present study is at odds with prior reports in other species, including, human, rat, and rabbit corneas (Lambiase et al., 2000b; Lambiase et al., 2002). It is possible that under resting physiological conditions dog keratocytes contain levels of NGF
and trkA that are below the limits of immunodetection. The presence in the same tissue sections of positively stained corneal epithelial cells and stromal nerves argues against a deficiency of the immunohistochemical method. The NGF- and trkA-IR nerve fibers seen here in the dog cornea are morphologically indistinguishable from
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Fig. 6. Corneal epithelium NGF content 1 week after corneal epithelial debridement. Note the increase of NGF in the epithelium of wounded corneas. *pZ0.024 comparing the value to baseline; !pZ0.029 comparing the value to the unwounded contralateral eyes.
Fig. 7. Lacrimal tissue NGF content 1 week after corneal epithelial debridement. Note that NGF concentrations are higher than unwounded controls both ipsilateral and contralateral to the wounded side. *p!0.001 comparing the value to normal values; **p!0.0001 comparing the value to normal values.
the canine corneal nerve fibers described in our earlier study (Marfurt et al., 2001). The source of the NGF contained within these nerves is a matter of speculation. Corneal nerves supply a rich innervation to the corneal epithelium and perhaps NGF released by the epithelial cells is internalized by the nerve terminals and transported retrogradely into the axons. Alternatively, trigeminal ganglion neurons are able to produce endogenous NGF and trkA and can transport these proteins into their peripheral axons (Jacobs and Miller, 1999). The simultaneous demonstration within the cornea of NGF- and trkA immunoreactive nerves
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and epithelial cells suggests intriguing possibilities for mutual trophic interactions. For example, epitheliumderived NGF may promote peripheral nerve maintenance, regenerative sprouting, and expression of trophic neuropeptides such as substance P and CGRP (MacLean et al., 1988; Donnerer et al., 1992), while NGF released from corneal nerves may promote epithelial homeostasis and reparative processes associated with wound healing. The results of the present study have demonstrated, by both ELISA and immunohistochemistry, the presence of NGF in normal dog lacrimal and third eyelid glands and corroborates an earlier report of NGF mRNA in the human lacrimal gland (Nguyen et al., 1997). In fact, levels of NGF were highest in lacrimal glands and glands of the third eyelid. Combined with the presence of NGF in the tear film, this data suggests that lacrimal tissue is an important source of NGF in vivo. Analogous patterns of NGF and trkA immunostaining suggests that individual acinar cells probably express both proteins and that lacrimal NGF likely acts in an autocrine or paracrine fashion to modulate gland activity. The immunohistochemical data presented here reveals that individual acinar cells in the same secretory unit exhibit substantial cell-to-cell differences in resting levels of NGF and trkA expression and/or storage. Because acinar cell secretion is under the control of the parasympathetic nervous system, the cell-to-cell differences in acinar protein expression seen here may reflect distributional or functional differences in parasympathetic innervation (Williams et al., 1994). Finally, the current study has shown that NGF is present in normal canine tears, confirming similar observations from tear fluids in human subjects (Vesaluoma et al., 2000). The results of the ELISA and immunohistochemical studies strongly suggest that tear fluid NGF is contributed from multiple sources, including, lacrimal and third eyelid glands, corneal epithelium and perhaps corneal and conjunctival nerves. The demonstration of NGF in canine tear fluid under resting physiologic conditions suggests that NGF, at appropriate concentrations and perhaps in synergism with other growth factors in the tear film, plays an important role in corneal epithelial maintenance and homeostasis.
Table 2 MeanGSD epithelial healing rates and time to healing after corneal epithelial debridement Treatments
Murine NGF (nZ6) Rh NGFc (nZ4) Anti-NGF Ab. (nZ6) a b c
Healing ratea (mm hrK1)
Time to heal (hr)
Treated
Controlb
p value
Treated
Controlb
p value
67.9G13.1 62.3G17.9 62.8G16.8
61.2G12.8 64.8G13.5 64.6G12.4
0.40 0.46 0.64
53.8G7.2 60.6G4.6 64.7G8.9
59.5G14.5 56.2G8.3 56.8G7.4
0.40 0.18 0.12
The rates were calculated by the decrease in wound radius. Treated with bovine serum albumin. Recombinant human Nerve Growth Factor.
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4.2. Alterations in corneal NGF and trkA expression following epithelial wounding Unilateral corneal epithelial debridement produced a significant ipsilateral increase in canine epithelial NGF expression that was demonstrated in the present study by two independent methods, i.e. immunohistochemistry (2 days post-wounding) and ELISA (7 days post-wounding). These findings confirm previous reports of increased epithelial NGF expression and/or binding in rats after corneal epithelial debridement (Lambiase et al., 2000b) and after skin wounding (Constantinou et al., 1994; Matsuda et al., 1998). The simultaneous increases in NGF and trkA expression seen here in the dog cornea after wounding occur preferentially in corneal epithelial cells near the wound margin and throughout the basal epithelium. This pattern of NGF/trkA upregulation is notable because it occurs primarily in cells that are actively engaged in reparative behaviors such as migration and proliferation. It is tempting to speculate that by augmenting activity in the endogenous NGF/trkA autocrine-paracrine system, migratory and proliferating corneal epithelial cells can further increase their responsivity to the wound-promoting effects of NGF. In the present study, epithelial scrape injury also produced, as revealed by immunohistochemical analysis 48 hr later, a widespread and pronounced upregulation of NGF and trkA expression in stromal keratocytes. One of the first responses to corneal epithelial debridement is the loss of keratocytes from the underlying stroma (Wilson et al., 1996; Zhao et al., 2001) and in the current study the presence of a largely ‘cell-free’ zone beneath the epithelial injury site is confirmed, although small numbers of scattered keratocytes persist. Whether the latter cells are resident keratocytes that fail to degenerate, or keratocytes that have migrated into the area in the past 48 hr, is unknown. Regardless, these keratocytes, and virtually every keratocyte in the corneal stroma outside the cell-poor zone, stain prominently for NGF and/or trkA. The sheer numbers of keratocytes that express either NGF or trkA in serial tissue sections strongly suggests that most, if not all, keratocytes express both proteins after wounding. It is tempting to speculate that keratocytes may utilize this NGF/trkA autocrine-paracrine system to promote migration and proliferation and thereby facilitate repopulation of the cell-poor zone. Future work should examine alterations in keratocye apoptosis by NGF following epithelial injury. 4.3. NGF alterations in the lacrimal gland and tears Following corneal epithelial scrape injuries, the lacrimal gland increases its production of several growth factors, including, EGF, HGF, TGF-b and KGF (Wilson et al., 1999; Lim et al., 2003). The results of the present ELISA study adds to this list by showing that NGF production is also increased, not only in the lacrimal gland but also in the third eyelid gland. The mechanism by which corneal epithelial
injury stimulates lacrimal gland NGF production is uncertain, but most likely involves a reflex neural circuit comprised of corneal sensory nerves, brainstem connections, and lacrimal parasympathetic (cholinergic) innervation. Cholinergic stimulation also increases EGF secretion by the lacrimal gland (Wilson et al., 1991). Unilateral wounding of the dog cornea also causes NGF concentrations to increase in the contralateral lacrimal gland, third eyelid gland, and tear fluid. The contralateral response may also be mediated neuronally (through crossed brainstem connections), or perhaps systemically. In support of the latter hypothesis, it has been shown that skin wounding in mice causes the salivary glands to release NGF into the systemic circulation and increases serum NGF levels (Matsuda et al., 1998). The rapid and significant increases in lacrimal gland and tear NGF levels seen here after corneal epithelial wounding provide further support for the concept of the ocular surface as a ‘functional unit.’ According to this model, corneal epithelial maintenance and wound healing depend on the coordinated biological activities of the cornea, tear film, main and accessory lacrimal glands, and their interconnecting reflex neural circuits (Stern et al., 1998; Mathers, 2000). Furthermore, the abrupt decrease of NGF at 48 hr, when the wounds were almost healed, may indicate a downregulation once the epithelium is reformed. 4.4. Topical NGF and corneal wound healing Neither topical NGF nor topical anti-NGF blocking antibody altered the corneal epithelial wound healing rate when applied to otherwise healthy dog eyes in this study. These findings contrast with previous reports from another laboratory. Lambiase and coworkers reported that topical application of murine NGF accelerated corneal epithelial wound healing, and anti-NGF antibody attenuated wound healing, in the rabbit cornea (Lambiase et al., 2000b). In clinical studies by these same authors, topical NGF was reported to accelerate the healing of corneal ulcers in patients with neurotrophic keratitis from various causes (Lambiase et al., 1998; Bonini et al., 2000). The reasons for the apparent discrepancy between our data and those of Lambiase et al. are not clear, but several factors may contribute to the differences. It is possible that endogenous NGF concentrations at the wound site in normal, control dogs after corneal wounding may already be optimal and thus supplementation through topical application would not improve the wound healing response. In contrast, the positive responses to topically applied NGF observed in human corneas occurred in patients with preexisting epithelial defects and ocular surface environments that may have been deficient in one or more essential growth factors (Lambiase et al., 1998; Bonini et al., 2000). Another possible contributing factor is the relative specificity of the agents used. It is possible that the murine 2.5S NGF, rh NGF and blocking antibody used in this study do
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not bind with the same avidity to canine receptors, or that intracellular pathways activated subsequent to binding are different. NGF with different forms has been isolated from various sources and methods including 2.5S NGF with 26 kDa, and 7S NGF with 14 or 116 kDa from mouse salivary, submaxillary, or submandibular gland, respectively (Young et al., 1978; Shooter, 2001). The murine NGF used in this study has been shown to act on other species such as mice, rats, rabbits and humans (Li et al., 1980; Kruse and Tseng, 1993; Lambiase et al., 2000b); however, species variability in response to NGF from different sources have also been reported. Exogenous 7S NGF and HMW-NGF obtained from mouse salivary gland affected skin wound healing differently when tested in the Syrian hamster and mouse (Li et al., 1980). Collectively, these data suggest that the wound healing properties of topical NGF may exhibit significant species-specific differences. The results of the current study have shown that NGF is present in measurable quantities in normal dog tears, corneal epithelium, lacrimal gland, and third eyelid gland. Furthermore, NGF concentrations in these tissues are significantly increased, either ipsilaterally or bilaterally, following unilateral corneal epithelial wounding. Application of exogenous NGF, however, does not alter wound healing in vivo in the dog.
Acknowledgements This work was supported by a grant from the National Eye Institute (EY 10841-01) and was presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology in Fort Lauderdale, Florida, May 2001.
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