Four full-thickness skin wounds made in normal mice led to the significant increase in levels of
nerve growth factor (NGF) in sera and in wounded skin tissues. Since sialoadenectomy before the wounds inhibited the rise in serum levels of NGF, the NGF may be released from the salivary gland into the blood stream after the wounds. In contrast, the fact that messenger RNA
and protein of NGF were detected in newly formed epithelial cells at the edge of the wound
and fibroblasts consistent with the granulation tissue produced in the wound space, suggests
that NGF was also produced at the wounded skin site. Topical application of NGF into the
wounds accelerated the rate of wound healing in normal mice and in healing-impaired diabetic
KK/Ta mice. This clinical effect of NGF was evaluated by histological examination; the increases in the degree of reepithelialization, the thickness of the granulation tissue, and the density of extracellular matrix were observed. NGF also increased the breaking strength of healing
linear wounds in normal and diabetic mice. These findings suggested that NGF immediately
and constitutively released in response to cutaneous injury may contribute to wound healing
through broader biological activities, and NGF improved the diabetic impaired response of wound healing.
 |
Introduction |
Repair of wounds is a chain of processes necessary for
removal of damaged tissues or invaded pathogens
from the body and for complete or incomplete remodeling
of injured tissues. The healing process requires a sophisticated interaction between inflammatory cells, biochemical
mediators including growth factors, extracellular matrix molecules, and microenvironmental cell populations (1, 2). Inflammatory cells, keratinocytes, and fibroblasts in the wound
space and border produce and release a variety of growth factors such as platelet-derived growth factor (PDGF)1, epidermal growth factor (EGF), fibroblast growth factor (FGF), and TGF, which have biological activities to stimulate infiltration of inflammatory cells into the wound space, induce proliferation of keratinocytes and fibroblasts, lead to
new formation of capillaries in the granulation tissue, and
modulate extracellular matrix deposition and reconstitution
of the injured area (1). In fact, topical application of
some growth factors is successful to accelerate healing of
full-thickness wounds in normal mice and normalize a delayed healing response of diabetic mice (6, 7).
Cutaneous wounds often cause anatomical and/or functional damage of peripheral sensory neurons widely distributed in the skin, and nerve growth factor (NGF), which is
probably produced in the affected tissue area, may be essential to regenerate the injured neurons. Patients with diabetes mellitus manifest acute and chronic complications including cutaneous infections, immunodisturbance, and vascular
and neuropathic dysfunctions (8). Impaired production of
NGF has been reported in the submaximal gland of genetically diabetic db/db mice (9) and streptozotocin-induced
diabetic mice (10), and in the serum and skin of patients
with diabetes mellitus (11, 12). Although NGF is a neurotrophic polypeptide mandatory for the development and
function of peripheral and central neurons (13), recent
findings have shown that NGF regulates immune and inflammatory responses through direct and/or indirect effects
on immunocompetent cells (16). Biologic actions of
NGF are mediated through two types of specific receptors
with distinct affinities (23, 24); the low affinity receptor is a
75-kD glycoprotein and the high affinity receptor is a 140-kD
molecules with a transmembrane tyrosine kinase domain
that is coded by the trk protooncogene (25). We have been
studying novel roles of NGF in the processes of inflammation and tissue repair. NGF caused a significant stimulation
of granulocyte differentiation from human peripheral blood
and murine bone marrow cells (26), suppressed apoptosis of rodent neutrophils and peritoneal mast cells (29, 30),
enhanced functional properties of murine neutrophils and human eosinophils (20), and not only promoted colony
formation of murine IL-3-dependent bone marrow-derived
cultured mast cells, but also induced the phenotypic change
to connective tissue-type mast cells (31).
NGF is produced by many types of cells including fibroblasts (31, 32), keratinocytes (33), mast cells (34), and T cells (35). Therefore, there is a possibility that NGF produced at the wounded site may regulate the healing of the
cutaneous wounds. In the present study, we demonstrated
that cutaneous wounds resulted in NGF production by the
salivary gland and regenerated keratinocytes at the edge of
the wound and fibroblasts in the granulation tissue during a
wound healing process, and that the topical application of
NGF to cutaneous wounds accelerated the rate of wound
healing in normal and diabetic mice.
 |
Materials and Methods |
Mice.
SJL/J mice were provided from N. Watanabe (Jikei
University School of Medicine, Tokyo, Japan). C57BL/6 and genetically diabetic KK/Ta mice were purchased from Clea Japan
(Tokyo, Japan). All mice (male, 8-10 wk of age) were kept
within a filter-air laminar flow enclosure, and provided with standard laboratory food and water ad libitum. All KK/Ta mice were
diagnosed to be diabetic at the beginning of the study.
Cytokines and Other Reagents.
2.5S NGF isolated from murine submaxillary glands was a gift from A.M. Stanisz and J. Bienenstock (McMaster University, Hamilton, Ontario, Canada; reference 26). The preparations were purified by gel filtration on a
Sephadex G-75 column to remove traces of renin and IgG sometimes found as contaminants in the original preparation, and further purified by affinity column chromatography with anti-
mouse NGF mAb (clone
1). The affinity-purified NGF preparation was eluted as a single protein on an HPLC column (TSK 3,000; Beckman Instruments, Fullerton, CA) with a retention
time that corresponded to 27 kD (2.5S NGF dimer; reference
26). Neither EGF activity by an ELISA nor endotoxin activity by
a limulus assay was detected, even at a high concentration (10 µg/ml) of the ultrapurified NGF preparation. Neurotrophic activity of the ultrapurified NGF preparation was determined as
previously described (31). Recombinant murine IL-1
, IFN-
, and
TNF-
, and recombinant human PDGF B chain (PDGF-BB) homodimer were purchased from Genzyme Corp. (Cambridge, MA).
Recombinant human basic FGF (bFGF) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). EGF purified from
murine submaxillary glands was provided by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Recombinant human TGF-
1
was a gift from H. Akiyama (Kirin Brewery Company, Ltd., Tokyo, Japan). Rabbit anti-mouse 2.5S NGF polyclonal Ab and
goat anti-rabbit IgG (H+L) polyclonal Ab conjugated with peroxidase were obtained from Sigma Chemical Co. (St. Louis, MO)
and BioMakor (Kirat Weizmann, Rehovot, Israel), respectively.
Mouse anti-2.5S NGF mAb was purchased from Boehringer
Mannheim GmbH (Mannheim, Germany). All chemicals used were purchased from Sigma Chemical Co., unless otherwise indicated.
Surgical Wounding.
Mice were wounded by using a modification of the technique described by Denon et al. (36). Under pentobarbital sodium anesthesia, hairs on the dorsum of mice were
clipped, and four full-thickness round skin wounds (5 mm diam)
were prepared using a disposable skin punch equipment (Maruho
Co., Ltd., Osaka, Japan). Each wound was separated by at least
1.5 cm of unwounded skin. A group of SJL/J mice was sialoadenectomized or sham operated under pentobarbital sodium anesthesia 4 wk before wounding. Heparinized peripheral blood
was collected from the axillary artery at 0, 1, 3, 6, and 24 h after
the skin punching. Small pieces of skin samples were removed
from the wounds and normal dorsal sites on 0, 1, 3, 7, 10, and 14 d
after the skin punching. All the blood and skin samples were obtained under ether anesthesia, and were treated with protease inhibitors (Boehringer Mannheim GmbH) according to the manufacturer's instructions. NGF levels in sera and extracts from the
skin tissues were measured by an ELISA using anti-NGF mAb
(31), which was sensitive to a lower limit of 50 pg/ml.
Immunohistochemical Examination.
Small pieces were cut from
skin tissues with wounds gently smoothed and flattened onto a
piece of thick filter, and were fixed with 4% paraformaldehyde in
0.1 M phosphate buffer (pH 7.4) for 12 h at 4°C. Skin tissues
were embedded in paraffin and cut at 4 µm; the sections were
placed on silane-coated glass slides. Tissues were deparaffinized,
rehydrated, and washed in PBS (pH 7.4). After pretreatment with
a solution of PBS supplemented with 0.3% hydrogen peroxide
and 0.1% sodium azide for 10 min to inhibit endogenous peroxidase, the preparations were washed in PBS twice, and then incubated with blocking medium (10% normal goat serum in PBS)
for 10 min. Rabbit anti-mouse 2.5S NGF polyclonal Ab diluted 1:2,000 in PBS supplemented with 1% BSA was applied for overnight at 4°C. After washing in PBS twice, peroxidase-conjugated
goat anti-rabbit IgG (H+L) Ab diluted 1:100 in PBS was overlaid for 30 min. Visualization of the reaction products was performed with 0.2 mg/ml 3-3
diaminobenzidine tetrahydrochloride in PBS supplemented with 0.003% hydrogen peroxide. The
tissues were counterstained with hematoxylin after the immunoreactions. Thin sections of submaxillary glands were provided
as a positive control.
In Situ Hybridization.
A digoxigenin-labeled antisense oligonucleotide primer (5
-AAGGGAATGCTGAAGTTTAGTCCAGTGGGCTTCAGGGACAGAGTCTCC-3
) complementary to nucleotides 378-425 of messenger RNA (mRNA) of mouse
NGF (37) was commercially synthesized (Nippon Flour Mills
Corp., Tokyo, Japan). Deparaffinized tissue sections were washed
in 2× SSC for 10 min at 60°C, rinsed in 0.05M Tris-HCl (pH
7.6), and incubated in 100 µg/ml proteinase K (Nacalai Tesque,
Kyoto, Japan) in 0.05 M Tris-HCl (pH 7.6) for 10 min at 37°C.
After washing in PBS, the tissue preparations were immersed in
0.4% paraformaldehyde in PBS, pH 7.4, for 10 min at 4°C to arrest proteolytic activity of proteinase K and rinsed in water. To
inhibit endogenous alkaline phosphatase activity, the specimens
were treated with 0.2 N hydrogen chloride for 10 min. Hybridization was performed using a slight modification of the method
reported previously (38). The specimens were hybridized with
the digoxigenin-labeled probe (20 ng/ml) in a solution of 50%
formamide, 4× SSC, 0.02% Ficoll (type 400), 0.02% polyvinylpyrrolidone, 0.02% BSA, 0.5 µg/ml salmon sperm DNA, 1%
sarcosyl (N-lauroyl sarcosine), 10% dextran sulfate, 0.1 M phosphate buffer (pH 7.2), and 0.05 M dithiothreitol, for 16 h at 42°C
in a humidified chamber. After washing, the slides were incubated with alkaline phosphatase-conjugated sheep antidigoxigenin Ab (Boehringer Mannheim GmbH), and the reaction products were visualized according to the manufacturer's instructions.
A synthesized sense oligonucleotide primer was used as a negative control. The other control experiments were performed as follows: (a) RNase A treatment before hybridization, and (b) neither a probe nor antidigoxigenin Ab. In these experiments, little
or no positive reaction was detected.
Production of NGF by Fibroblasts and Keratinocytes.
The contact-inhibited Swiss albino mouse embryo-derived 3T3 fibroblasts,
obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan), and the transformed BALB/c mouse-derived keratinocytes (PAM 212), provided from I. Katayama (Tokyo Medical and Dental University School of Medicine, Tokyo, Japan), were seeded in 35-mm culture dishes (Nunc, Roskilde, Denmark) at a concentration of 5.0 × 104 cells/ml in 1 ml of
-MEM (GIBCO
BRL, Gaithersburg, MD) with 10% FCS (Hyclone Labs, Logan,
UT). The culture dishes that contained a confluent monolayer of
3T3 fibroblasts (0.5-1.5 × 106 cells) on 3 d in culture were further incubated at 37°C for 6 d in humidified atmosphere with 5%
CO2 in air after the culture medium was replaced with 1 ml of
fresh medium containing various concentrations of cytokines or
histamine. PAM 212 keratinocytes were incubated with various
concentrations of cytokines or histamine for 2 d. The culture medium of both the cells was collected to measure NGF levels (see
above) and the number of fibroblasts and keratinocytes was
counted in trypsinized cultures.
Treatment of Wound.
Two different types of experiments were
conducted to evaluate a stimulating effect of NGF on wound
healing in C57BL/6 and genetically diabetic KK/Ta mice (39-
41). First, after skin punching (see above), 20 µl (1 µg) of 2.5S
NGF and vehicle solution alone (
-MEM with 10% FCS) were
daily applied to two left and two right wounds, respectively. Applications were carried out under pentobarbital sodium anesthesia
until the third day, and a healing term was assessed macroscopically. In other experiments, the wounds were removed 8 d after
skin punching, and were fixed in phosphate-buffered formalin.
Paraffin sections (5 µm thick) were made by routine methods and
stained with hematoxylin and eosin. Second, breaking strength of
healed linear wounds was examined according to a slight modification of the methods previously reported (6). An anterior-posterior linear incision (4 cm in length) in full thickness was applied to the dorsum of mice with a scalpel under pentobarbital sodium anesthesia. 50 µl (2 µg) of 2.5S NGF or vehicle solution alone were administrated to the incisions, and then the wounds were closed by wound clips placed at 1-cm intervals. Mice were killed 9 d later, and three pieces of skin (0.8 cm in width) between the
wound clips were cut vertical to the linear incision. Breaking strength
of wounds was measured by using a Rheo meter (NRM-2002J; Fudoh Kogyo Co., Ltd, Tokyo, Japan). The ends of the skin strip were pulled at a constant speed (20 cm/min), and breaking
strength was expressed as the mean maximum level of tensile
strength (g/mm) before separation of wounds.
Statistical Analysis.
Two-tailed Student's t test was done for
statistical analysis of the data and P <0.05 was taken as the level of
significance.
 |
Results |
NGF Levels in Serum.
Four full-thickness round wounds
were made at the dorsal skin in SJL/J mice, and serum samples were collected under ether anesthesia at various times
after the skin punching. Although no NGF was detected in
sera isolated from anesthetized mice before the skin punching, the cutaneous wounding resulted in a rapid increase in
serum levels of NGF. Serum NGF was at a significant level
of 0.72 ng/ml at 1 h, reached a maximal level of ~5.20 ng/ ml at 6 h, and retained a significant level of ~0.88 ng/ml
even 24 h after the skin punching (Fig. 1). The serum collected at 6 h after the skin punching stimulated the outgrowth of nerve fibers from rat pheochromocytoma cells
(PC12); the neurotrophic activity was completely abolished
by the addition of anti-NGF Ab (data not shown).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1.
Serum NGF levels
in male SJL/J mice after cutaneous wounding. Each point represents the mean ± SE of three
separate experiments using duplicate samples.
|
|
NGF Levels in Skin Tissues.
To examine the possible local
production of NGF at wounded sites, NGF contents at unwounded and wounded sites were measured on various
days after skin punching. All the cutaneous wounds were
completely closed by 11 d. Low levels of NGF were detected at uninjured control skin sites isolated on various
days after wounding, ranging from 0.81 to 1.7 ng/g. In
contrast, at the wounded sites, NGF reached a maximal
level of 7.8 ng/g 1 d later, and then its levels were gradually
decreased but were higher than those at uninjured control
skin sites during the period of 14 d (Fig. 2).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
NGF levels at the
wounded skin site (closed) and
control unwounded skin site
(open) of male SJL/J mice after
cutaneous wounding. Each point
represents the mean ± SE of
three separate experiments using
duplicate samples.
|
|
Effect of Sialoadenectomy on NGF Levels in Sera and Wounded
Skin Sites.
Since the increased NGF in sera after fighting
stress has been reported to be originated from the submaxillary glands (42), we conducted some experiments to examine
whether sialoadenectomy before cutaneous wounds affected
NGF levels in sera and wounded skin sites. Serum NGF
levels at 6 h in sialoadenectomized SJL/J mice were lower
than the detection limit of an ELISA, but the increased levels were observed in sera of sham-operated mice as roughly
comparable to those in nonoperated normal mice (Table 1).
On the other hand, sialoadenectomy had no influence on
NGF contents at wounded skin sites 3 d after the skin
punching. Thus, we concluded that cutaneous wounds led
to rapid release of NGF from the submaxillary glands to the
peripheral blood and the subsequent local production of
NGF at the wounds.
Production of NGF at Wounded Skin Sites.
To identify cell
populations that produced NGF at wounded skin sites in
SJL/J mice 3 d after skin punching, in situ hybridization analysis and immunohistochemical examination were provided. Granulation tissue including fibroblasts, capillaries,
and various kinds of inflammatory cells filled the wound
space under crustal tissue. Epithelium at the edge of the
wound was several cell layers thick. The leading single cell
layer edge of the epithelium was evident over the newly
formed granulation tissue, but reepithelialization was only
10-20% at this time. The stratified epithelial cells at the
wound edge showed positive staining for mRNA and protein of NGF; deeper layer epithelial cells were strongly
positive for its mRNA staining, but superficial layer epithelial cells were strongly positive for its protein staining (Fig.
3, B and F). In addition to the neoepithelium, fibroblasts in
granulation tissue formed in the wound space and wound
edge were positive for mRNA and protein of NGF (Fig. 3,
D and H). In contrast, little or no reaction was observed in
epidermal keratinocytes and dermal fibroblasts at the unwounded control skin site. Thus, NGF was produced by stratified epithelial cells and fibroblasts in granulation tissue formed after wounding.

View larger version (125K):
[in this window]
[in a new window]
|
Fig. 3.
Cellular localization of mRNA and protein of NGF in newly
formed epithelium at edge of the wound (B and F, original magnification: 240) and granulation tissue produced in the wound space (D and H, original magnification: 550). All specimens were obtained from male SJL/J
mice 3 d after cutaneous wounding. Basal cells of the epidermis show
positive reaction for mRNA expression of NGF (B) and superficial epithelial cells show positive reaction for protein of NGF (F). Positive reaction for both the mRNA (D) and protein (H) of NGF is observed in fibroblasts in the granulation tissue. No positive reactions are observed in
the sections treated with the sense primer (A and C) and the irrelevant Ab
instead of anti-NGF Ab (E and G).
|
|
Effect of Various Cytokines and Histamine on NGF Production by Fibroblasts and Keratinocytes.
Since NGF is produced
and released from mouse fibroblasts and keratinocytes in
vitro (31, 33), we examined the effect of inflammatory cytokines (EGF, bFGF, IFN-
, IL-1
, PDGF-BB, TGF-
1, and TNF-
) and histamine on NGF production by 3T3 fibroblasts and PAM 212 keratinocytes. NGF levels were assessed by an ELISA in the culture medium collected from
3T3 fibroblasts on 6 d in culture and PAM 212 cells on 2 d
in culture. When 3T3 fibroblasts and PAM 212 cells were
cultured in medium alone, the collected culture medium
contained 88 ± 3 pg/ml and 111 ± 4 pg/ml, respectively. The addition of EGF, bFGF, IFN-
, IL-1
, PDGF-BB,
TGF-
1, or histamine to 3T3 fibroblasts increased NGF
levels in the medium at the individual optimal doses by
more than three times as compared with medium alone
(Fig. 4). In contrast, the addition of TNF-
at doses of 0.2-2
ng/ml resulted in slight enhancement of NGF production
(Fig. 4). When PAM 212 cells were cultured with each cytokine or histamine, NGF levels were 1.5-2.5-fold higher
than those in medium alone at the individual optimal doses
of all the agents (Fig. 5). There was no significant difference in the number of fibroblasts and keratinocytes at the
end of the culture between individual groups.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Production of NGF by 3T3 fibroblasts stimulated with various cytokines and histamine. NGF levels in culture medium were measured by a sandwich ELISA as described in Materials and Methods. Each point represents the mean ± SE of three separate experiments using duplicate samples.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Production of NGF by PAM 212 keratinocytes stimulated
with various cytokines and histamine. NGF levels in culture medium
were measured by a sandwich ELISA as described in Materials and Methods. Each point represents the mean ± SE of three separate experiments using duplicate samples.
|
|
Effect of NGF on the Rate of Wound Healing.
We attempted
to assess a possible positive effect of NGF on the rate of cutaneous wound healing in C57BL/6 control mice and genetically diabetic KK/Ta mice. In C57BL/6 mice, right
full-thickness round wounds topically treated with only
medium alone for 3 d were closed by 11 d (Fig. 6). In KK/
Ta mice, on the other hand, wound closure was delayed
>7 d compared to that in C57BL/6 mice, indicating that
the rate of wound healing was impaired in KK/Ta mice (P
<0.001), the same as genetically diabetic db/db mice (6). When 1 µg NGF was applied to left wounds once per day
for 3 d beginning with the day of skin punching, the rate of
wound healing was significantly accelerated in both
C57BL/6 and KK/Ta mice (Fig. 6). Next, histological examination was performed on the wounds 8 d later. In
C57BL/6 mice, topical application of NGF led to the slight
accelerating effect on wound healing parameters: complete reepithelialization, an increase in the degree of matrix density, and decreased infiltration of neutrophils (Table 2 and
Fig. 7). On the other hand, in KK/Ta mice an impairment
in wound healing was evident in incomplete reepithelialization, low deposition of extracellular matrix, and continuous infiltration of numerous neutrophils, but the topical
application of NGF improved the parameters of wound healing, which were comparable to those in control C57BL/6 mice without the application of NGF (Table 2 and Fig. 7).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 6.
Wound healing accelerated by topical applications
of NGF in control C57BL/6
mice and diabetic KK/Ta mice.
NGF (1 µg) or vehicle solution
alone was applied to the wound
space each day for 3 d beginning
with the day of wounding as described in Materials and Methods, and a healing term was examined macroscopically. Each histogram
represents the mean ± SE of six mice per group. *P <0.02; P <0.01;
when compared with diluent alone.
|
|

View larger version (134K):
[in this window]
[in a new window]
|
Fig. 7.
Histological features of wound specimens from control C57BL/6 mice (A and B) and diabetic KK/Ta mice (C and D) 8 d after cutaneous wounding. NGF (1 µg; B and D) or vehicle solution alone (A and C) was applied to the wound space each day for 3 d beginning with the day of wounding as described in Materials and Methods. Sections were stained with hematoxylin and eosin. Original magnification: 60. Arrow heads, the original wound
margin.
|
|
To further evaluate the effect of NGF on wound healing, we attempted to measure breaking strength of anterior-posterior incisional wounds treated with 2 µg NGF or
treated with diluent solution alone. Wound specimens were
obtained from C57BL/6 and KK/Ta mice 9 d after wounding. Fig. 8 shows that breaking strength of wounds treated
with diluent solution alone in C57BL/6 was larger than wounds in KK/Ta mice (P <0.01). A single dose of treatment with NGF was sufficient to induce a significant increase in breaking strength by >1.5 and 2 times in C57BL/6
and KK/Ta mice, respectively. Breaking strength of wounds
treated with NGF in KK/Ta mice was comparable to that
of wounds treated with diluent solution alone in C57BL/6
mice.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of NGF on
wound tear strength in control
C57BL/6 mice and diabetic KK/
Ta mice. NGF (2 µg) or vehicle
solution alone was applied to the
wound space shortly after the
wounding, and wound tear
strength was measured as described in Materials and Methods. Each histogram represents the mean ± SE of four mice per group.
*P <0.01; when compared with diluent alone.
|
|
Specificity of NGF Effects on Wound Healing.
To determine
the specificity of the accelerating effects of NGF on wound
healing in diabetic KK/Ta mice, NGF was pretreated in
vitro with anti-NGF Ab before the topical application to
round wounds and linear incisions. As shown in Table 3,
NGF pretreated with control Ab induced to accelerate the
rate of the wound healing parameters. On the other hand,
pretreatment with anti-NGF Ab completely abolished the
wound-healing accelerating effects of NGF. Thus, we concluded that topical administration of NGF accelerated the rate of cutaneous wound healing in control normal mice
and healing-impaired diabetic mice.
 |
Discussion |
NGF has the potential to enhance survival and functions
of immunocompetent cells, such as neutrophils, eosinophils,
mast cells, macrophages, and lymphocytes in rodents (17,
18, 20, 21, 29) and humans (19, 22, 43), suggesting
a possible ability of NGF to promote the rate of cutaneous
wound healing. To clarify this point, the present study was
conducted. The full-thickness skin wounds at the dorsum
of normal mice were able to lead to the rapid increase in
levels of NGF in the peripheral blood that possessed a neurotrophic ability. This response was completely abolished
by the sialoadenectomy 4 wk before the cutaneous wounds, suggesting that biologically active NGF produced in the
submaxillary gland may be released into the peripheral blood.
This appears to be consistent with the result of Aloe et al.
(42) that showed that aggressive behavior induced the rapid
release of NGF from the salivary gland into the blood
stream through stimulation of the sympathetic nerve in
mice. Thus, biologically active NGF may be released from
the salivary gland into the blood stream in response to the
cutaneous wound as well as the fighting stress. We also demonstrated that the cutaneous wounds resulted in the
significant increase in levels of NGF in the affected cutaneous tissue after the rapid release of NGF into the peripheral
blood. Liu et al. (46) reported the increased NGF levels in a
wound chamber implanted in axotomized rat sciatic nerve,
whereas serum NGF levels remained low. Since the increased level of NGF was not influenced by the sialoadenectomy, and since mRNA and protein of NGF were observed in not only stratified epithelial cells at the edge of
the wound and also fibroblasts in the granulation tissue
produced in the wound space, the increased NGF in the
wounded skin may be mainly produced by newly formed
epithelial cells and fibroblasts in the granulation tissue.
A variety of inflammatory cytokines produced by local
tissues at the wound acts individually and/or collaboratively
in processes of the wound healing and tissue remodeling,
and synthesis of the cytokines also seems to be regulated
mutually. In fact, IL-1 has a potent ability to upregulate
synthesis of NGF in nonneuronal cells of injured rat sciatic
nerves (47) and in cultured rat fibroblasts (48). We attempted to demonstrate whether various cytokines (EGF,
bFGF, IFN-
, IL-1
, PDGF-BB, TGF-
1, and TNF-
) and histamine that are produced and released in injured tissues (1, 3), affect NGF production by both 3T3 fibroblasts and PAM 212 keratinocytes in vitro. Interestingly, all
of the reagents except for TNF-
enhanced NGF production of both of the cell lines. Since little or no reaction for
mRNA and protein of NGF was detected in epidermal keratinocytes and dermal fibroblasts at the uninjured control
skin site, NGF synthesis in both the cell populations activated and proliferated after the wounds seems to be regulated by such cytokines released from various cell components in local tissues including infiltrating inflammatory cells.
The local application of NGF into cutaneous wounds was
sufficient to accelerate the rate of wound healing in normal
mice and normalize the delayed healing response in diabetic KK/Ta mice. In addition to the results of healing rate
and breaking strength of wounds, the histological findings,
such as the increases in the degree of reepithelialization, the
thickness of the granulation tissue, and the density of extracellular matrix, provided distinct evidence that NGF had a
biological ability to improve the degree of the parameters
of wound healing in normal mice and even in healing-impaired diabetic mice. NGF also modulates proliferation of keratinocytes in mice (49) and humans (50) through the high affinity receptor, suggesting that NGF produced from
newly formed epithelial cells at the edge of the wound may
support reepithelialization through autocrine stimulation
mechanisms involving synergy with the other cytokines. In
contrast, since murine 3T3 fibroblasts have no expression
of the NGFR on their surface (21), the granulation tissue
and matrix formation induced after NGF application might be caused by indirect promoting effects through cytokines
such as bFGF, PDGF, and TGF-
1, which contribute to
proliferation of fibroblasts and synthesis of extracellular matrix by fibroblasts (51).
Peripheral neuropathy that occurs by complicated metabolic mechanisms is one of the common complications distressing patients with diabetes mellitus; impairment of sensory neurons presents as pain and loss of sensation, and
often results in cutaneous infection and impaired wound
healing (8). The recent report (13) demonstrating the decreased levels of endogenous NGF, a sensory neurotrophic
factor, and substance P, a sensory neurotransmitter, in the
skin of patients with diabetes mellitus, gives rise to a possibility that impairment of NGF production may also contribute to the pathogenesis of diabetic peripheral neuropathy.
In fact, diabetic KK/Ta mice showed impaired regeneration of nerve fibers after wounding (data not shown), and
the topical administration of NGF significantly accelerated
the regeneration of nerve fibers that were roughly comparable to that of control C57BL/6 mice. Apfel et al. (54)
demonstrated that exogenously administered NGF was capable of preventing the behavioral and biochemical manifestations of sensory neuropathy in streptozotocin-induced
diabetic rats. The present study clearly demonstrated that
the topical administration of NGF into the full-thickness
wounds by skin punching normalized the defect of diabetic
KK/Ta mice regarding the rate of wound healing. Thus,
we consider that NGF has a potentiality as a therapeutic agent for the normalization of the diabetic impaired response of wound healing.
Address correspondence to Hiroshi Matsuda, Department of Veterinary Clinic, Faculty of Agriculture, Tokyo
University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183, Japan. Phone: 81-423-67-5784; Fax: 81-423-60-8830; E-mail: hiro{at}cc.tuat.ac.jp
We would like to thank Drs. J. Bienenstock and A.M. Stanisz (McMaster University, Hamilton, Ontario,
Canada) for providing ultrapurified NGF, Dr. I. Katayama (Tokyo Medical and Dental University, Tokyo,
Japan) for providing PAM 212 keratinocytes, and Dr. H. Akiyama (Kirin Brewery Company, Ltd., Tokyo,
Japan) for supplying TGF-
1. We also thank Dr. R. Tsuboi (Juntendo University, Tokyo, Japan) for helpful
discussions.
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture, Japan, for
Recombinant Cytokine's Project provided by the Ministry of Agriculture, Forestry, and Fisheries, Japan
(RCP 1998-4120), and from Lydia O'Leary Memorial Foundation.
1.
| Clark, R.A.F. 1996. Wound repair: overview and general
considerations. In The Molecular and Cellular Biology of
Wound Repair. R.A.F. Clark, editor. Plenum Press, New
York. 3-50.
|
2.
|
Martin, P..
1997.
Wound healing-aiming for perfect skin regeneration.
Science.
276:
75-81
[Abstract/Free Full Text].
|
3.
|
Pierce, G.F., and
T.A. Mustoe.
1995.
Pharmacologic enhancement of wound healing.
Annu. Rev. Med.
46:
467-481
[Medline].
|
4.
|
Bennett, N.T., and
G.S. Schultz.
1993.
Growth factors and
wound healing: biochemical properties of growth factors and
their receptors.
Am. J. Surg.
165:
728-737
[Medline].
|
5.
|
Moulin, V..
1995.
Growth factors in skin wound healing.
Eur.
J. Cell Biol.
68:
1-7
[Medline].
|
6.
|
Tsuboi, R., and
D.B. Rifkin.
1991.
Recombinant basic fibroblast growth factor stimulates wound healing-impaired db/db
mice.
J. Exp. Med.
172:
245-251
[Abstract].
|
7.
|
Brown, R.E.,
M.P. Breeden, and
D.G. Greenhalgh.
1994.
PDGF and TGF-alpha act synergistically to improve wound
healing in the genetically diabetic mouse.
J. Surg. Res.
56:
562-570
[Medline].
|
8.
| Siegel, J. 1995. Diabetes mellitus. In Perspectives on Pathophysiology. L.-E.C. Copstead, editor. W.B. Saunders Company, Philadelphia. 826-845.
|
9.
|
Kasayama, S., and
T. Oka.
1989.
Impaired production of
nerve growth factor in the submandibular gland of diabetic
mice.
Am. J. Physiol.
257:
E400-E404
[Abstract/Free Full Text].
|
10.
|
Ordonez, G.,
A. Fernandez,
R. Perez, and
J. Sotelo.
1994.
Low contents of nerve growth factor in serum and submaxillary gland of diabetic mice. A possible etiological element of
diabetic neuropathy.
J. Neurol. Sci.
121:
163-166
[Medline].
|
11.
|
Faradji, V., and
J. Sotelo.
1990.
Low serum levels of nerve
growth factor in diabetic neuropathy.
Acta Neurol. Scand.
81:
402-406
[Medline].
|
12.
|
Anand, P..
1996.
Neurotrophins and peripheral neuropathy.
Philos. Trans. R. Soc. Lond. B Biol. Sci.
351:
449-454
[Medline].
|
13.
|
Levi-Montalcini, R., and
P.U. Angeletti.
1963.
Essential role
of the nerve growth factor on the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells
in vitro.
Dev. Biol.
7:
653-659
.
|
14.
|
Thoenen, H., and
D. Edgar.
1985.
Neurotrophic factors.
Science.
229:
238-242
[Medline].
|
15.
|
Korsching, S..
1986.
The role of nerve growth factor in the
CNS.
Trends Neurosci.
9:
570-577
.
|
16.
|
Gee, A.P.,
M.D.P. Boyle,
L. Munger,
M.J.P. Lawman, and
M. Young.
1983.
Nerve growth factor: stimulation of polymorphonuclear leukocyte chemotaxis in vitro.
Proc. Natl.
Acad. Sci. USA.
80:
7215-7218
[Abstract].
|
17.
|
Pearce, F.L., and
H.L. Thompson.
1986.
Some characteristics of
histamine secretion from rat peritoneal mast cells stimulated
with nerve growth factor.
J. Physiol. (Lond.).
372:
379-393
[Abstract].
|
18.
|
Thorpe, L.W., and
J.R. Perez-Polo.
1987.
The influence of
nerve growth factor on the in vitro proliferative response of
rat spleen lymphocytes.
J. Neurosci. Res.
18:
134-139
[Medline].
|
19.
|
Otten, U.,
P. Ehrhard, and
R. Peck.
1989.
Nerve growth
factor induces growth and differentiation of human B lymphocytes.
Proc. Natl. Acad. Sci. USA.
86:
10059-10063
[Abstract].
|
20.
|
Kannan, Y.,
H. Ushio,
H. Koyama,
M. Okada,
M. Oikawa,
T. Yoshihara,
M. Kaneko, and
H. Matsuda.
1991.
2.5S nerve
growth factor enhances survival, phagocytosis, and superoxide production of murine neutrophils.
Blood.
77:
1320-1325
[Abstract].
|
21.
|
Susaki, Y.,
S. Shimizu,
K. Katakura,
N. Watanabe,
K. Kawamoto,
M. Matsumoto,
M. Tsudzuki,
T. Furusaka,
Y. Kitamura, and
H. Matsuda.
1996.
Functional properties of murine macrophages promoted by nerve growth factor.
Blood.
88:
4630-4637
[Abstract/Free Full Text].
|
22.
|
Hamada, A.,
N. Watanabe,
H. Ohtomo, and
H. Matsuda.
1996.
Nerve growth factor enhances survival and cytotoxic
activity of human eosinophils.
Br. J. Haematol.
93:
299-302
[Medline].
|
23.
|
Sutter, A.,
R.J. Riopelle,
R.M. Harris-Warrick, and
E.M. Shooter.
1979.
Nerve growth factor receptors: characterization of two distinct classes of binding sites on chick embryo
sensory ganglia cells.
J. Biol. Chem.
254:
4972-4982
.
|
24.
|
Yanker, B.A., and
E.M. Shooter.
1982.
The biology and
mechanism of action of nerve growth factor.
Annu. Rev. Biochem.
51:
845-868
[Medline].
|
25.
|
Kaplan, D.R.,
B.L. Hempstead,
D. Martin-Zanca,
M.V. Chao, and
L.F. Parada.
1991.
The trk proto-oncogene product: a
signal transducing receptor for nerve growth factor.
Science.
252:
554-558
[Medline].
|
26.
|
Matsuda, H.,
M.D. Coughlin,
J. Bienenstock, and
J.A. Denburg.
1988.
Nerve growth factor promotes human hemopoietic colony growth and differentiation.
Proc. Natl. Acad. Sci.
USA.
85:
6508-6512
[Abstract].
|
27.
|
Matsuda, H.,
J. Switzer,
M.D. Coughlin,
J. Bienenstock, and
J.A. Denburg.
1988.
Human basophilic cell differentiation
promoted by 2.5S nerve growth factor.
Int. Arch. Allergy Immunol.
86:
453-457
.
|
28.
|
Kannan, Y.,
H. Matsuda,
H. Ushio,
K. Kawamoto, and
Y. Shimada.
1993.
Murine granulocyte-macrophage and mast
cell colony formation promoted by nerve growth factor.
Int. Arch. Allergy Immunol.
132:
362-367
.
|
29.
|
Kannan, Y.,
K. Usami,
M. Okada,
S. Shimizu, and
H. Matsuda.
1992.
Nerve growth factor suppresses apoptosis of murine neutrophils.
Biochem. Biophys. Res. Commun.
186:
1050-1056
[Medline].
|
30.
|
Kawamoto, K.,
T. Okada,
Y. Kannan,
H. Ushio,
M. Matsumoto, and
H. Matsuda.
1995.
Nerve growth factor prevents
apoptosis of rat peritoneal mast cells through the trk proto-oncogene receptor.
Blood.
86:
4638-4644
[Abstract/Free Full Text].
|
31.
|
Matsuda, H.,
Y. Kannan,
H. Ushio,
Y. Kiso,
T. Kanemoto,
H. Suzuki, and
Y. Kitamura.
1991.
Nerve growth factor induces development of connective tissue-type mast cells in
vitro from murine bone marrow cells.
J. Exp. Med.
174:
7-14
[Abstract].
|
32.
|
Young, M.,
J. Ofer,
M.H. Blanchard,
H. Asdourian,
H. Amos, and
B.G.W. Arnason.
1974.
Secretion of a nerve
growth factor by primary chick fibroblast cultures.
Science.
187:
361-362
.
|
33.
|
Tron, V.A.,
M.D. Coughlin,
D.E. Jang,
J. Stanisz, and
D.N. Sauder.
1990.
Expression and modulation of nerve growth
factor in murine keratinocytes (PAM 212).
J. Clin. Invest.
85:
1085-1089
[Medline].
|
34.
| Leon, A., A. Buriani, R. dal Toso, M. Fabris, S. Romanello,
L. Aloe, and R. Levi-Montalcini. 1994. Mast cells synthesize, store, and release nerve growth factor. Proc. Natl. Acad. Sci. USA. 91:3739-3743.
|
35.
|
Ehrhard, P.B.,
P. Erb,
U. Graumann, and
U. Otten.
1993.
Expression of nerve growth factor and nerve growth factor
receptor tyrosine kinase Trk in activated CD4-positive T-cell
clones.
Proc. Natl. Acad. Sci. USA.
90:
10984-10988
[Abstract].
|
36.
|
Denon, D.,
M.A. Kowatch, and
G.S. Roth.
1989.
Production of wound repair in old mice by local injection of macrophages.
Proc. Natl. Acad. Sci. USA.
86:
2018-2020
[Abstract].
|
37.
|
Scott, J.,
M. Selby,
M. Uredea,
M. Quiriga,
G.I. Bell, and
W.J. Rutter.
1983.
Isolation and nucleotide sequence of cDNA
encoding the precursor of mouse nerve growth factor.
Nature.
302:
538-540
[Medline].
|
38.
|
Humpel, C.,
E. Lindqvidst, and
L. Olson.
1993.
Detection of
nerve growth factor mRNA in rodent salivary glands with
digoxigenin- and 32P-labeled oligonucleotides: effects of castration and sympathectomy.
J. Histochem. Cytochem.
41:
703-708
[Abstract/Free Full Text].
|
39.
|
Nakamura, M., and
K. Yamada.
1967.
Studies on a diabetic
(KK) strain of the mouse.
Diabetologia.
3:
212-221
[Medline].
|
40.
|
Reddi, A.S., and
R.A. Camerini-Davalos.
1988.
Hereditary diabetes in the KK mouse: an overview.
Adv. Exp. Med. Biol.
246:
7-15
[Medline].
|
41.
|
Hasegawa, M.,
Y. Ogawa,
G. Katsuura,
H. Shintaku,
K. Hosoda, and
K. Nakano.
1996.
Regulation of obese gene expression in KK mice and congenic lethal yellow obese KKAy
mice.
Am. J. Physiol.
271:
E333-E339
[Abstract/Free Full Text].
|
42.
|
Aloe, L.,
E. Alleva,
A. Bohn, and
R. Levi-Montalcini.
1986.
Aggressive behavior induces release of nerve growth factor
from mouse salivary gland into the bloodstream.
Proc. Natl.
Acad. Sci. USA.
83:
6184-6187
[Abstract].
|
43.
|
Ehrhard, P.B.,
U. Ganter,
J. Bauer, and
U. Otten.
1993.
Expression of functional trk protooncogene in human monocytes.
Proc. Natl. Acad. Sci. USA.
90:
5423-5427
[Abstract].
|
44.
|
Bischoff, S., and
C.A. Dahinden.
1992.
Effect of nerve
growth factor on the release of inflammatory mediators by
mature human basophils.
Blood.
792:
2662-2669
.
|
45.
|
Torcia, M.,
L. Bracci-Laudiero,
M. Lusibello,
L. Nencioni,
D. Labardi,
A. Rubartelli,
F. Cozzolino,
L. Aloe, and
E. Garaci.
1996.
Nerve growth factor is an autocrine survival factor
for memory B lymphocytes.
Cell.
85:
345-356
[Medline].
|
46.
|
Liu, H.M.,
H.Y. Lei, and
K.P. Kao.
1995.
Correlation between NGF levels in wound chamber fluid and cytological
location of NGF and NGF receptor in axotomized rat sciatic
nerve.
Exp. Neurol.
132:
24-32
[Medline].
|
47.
|
Lindholm, D.,
R. Heumann,
M. Meyer, and
H. Thoenen.
1987.
Interleukin-1 regulates synthesis of nerve growth factor
in non-neuronal cells of rat sciatic nerve.
Nature.
330:
658-659
[Medline].
|
48.
|
Lindholm, D.,
R. Heumann,
B. Hengerer, and
H. Thoenen.
1988.
Interleukin 1 increases stability and transcription of
mRNA encoding nerve growth factor in cultured rat fibroblasts.
J. Biol. Chem.
263:
16348-16351
[Abstract/Free Full Text].
|
49.
|
Paus, R.,
M. Luftle, and
B.M. Czarnetzki.
1994.
Nerve growth factor modulates keratinocyte proliferation in murine
skin organ culture.
Br. J. Dermatol.
130:
174-180
[Medline].
|
50.
|
Di Marco, E.,
M. Mathor,
S. Bondanza,
N. Cutuli,
P.C. Marchisio,
R. Cancedda, and
M. De Luca.
1993.
Nerve growth
factor binds to normal human keratinocytes through high and
low affinity receptors and stimulates their growth by a novel
autocrine loop.
J. Biol. Chem.
268:
22838-22846
[Abstract/Free Full Text].
|
51.
|
Buckley-Sturrock, A.,
S.C. Woodward,
R.M. Senior,
G.L. Griffin,
M. Klagsbrun, and
J.M. Davidson.
1989.
Differential stimulation of collagenase and chemotactic activity in fibroblasts derived from rat wound repair tissue and human skin
by growth factors.
J. Cell. Physiol.
138:
70-78
[Medline].
|
52.
|
Pierce, G.F.,
T.A. Mustoe,
B.W. Altrock,
T.F. Deuel, and
A. Thomason.
1991.
Role of platelet-derived growth factor in
wound healing.
J. Cell. Biochem.
45:
319-326
[Medline].
|
53.
|
Roberts, A.B.,
M.B. Sporn,
R.K. Assoian,
J.M. Smith,
N.S. Roche,
L.M. Wakefield,
U.I. Heine,
L.A. Liotta,
V. Flalnga,
J.H. Kehrl, and
A.S. Fauci.
1986.
Transforming growth factor type B: rapid induction of fibrosis and angiogenesis in vivo
and stimulation of collagen formation in vitro.
Proc. Natl.
Acad. Sci. USA.
83:
4167-4171
[Abstract].
|
54.
|
Apfel, S.C.,
J.C. Arezzo,
M. Brownlee,
H. Federoff, and
J.A. Kessler.
1994.
Nerve growth factor administration protects
against experimental diabetic sensory neuropathy.
Brain Res.
634:
7-12
[Medline].
|