The effect of endothelial cell growth factor on peripheral nerve regeneration

The effect of endothelial cell growth factor on nerve regeneration

peripheral

MICHAEL S. SMITH,PhD,MD, and J. DALEBROWNE,MD, FACS,Winston-Salem, North Carolina Neural regeneration offer grafting can be unpredictable. In an effort to enhance the return of function after cable grafting, we studied the effects of an angiogenic factor, endothelial cell growth factor (ECGF), on regenerating nerves. Cable grafts on the sciatic nerve were established in 18 rats and treated with ECGF or a control saline solution. At 5 weeks, nerve conduction studies were performed, and the animals were killed for histologic measurements of graft vascularity and axon counts. A significant increase in vascularity was noted in the treated group versus the control group; neither the axon counts nor the nerve conduction velocities differed significantly between the two groups, although the treated group appeared to show improved neural conduction compared with the control group. (Otolaryngol Head Neck Surg 1998; 118:178-82.)

D u r i n g the past several years, many studies have evaluated the effects of various growth factors on tissue growth and healing. The effects of growth factors on nerve regeneration are of significant interest because the ability of nervous tissue to regenerate is relatively poor. Other than studies on vascularized nerve pedicle grafts, however, very little has been written on growth factors and their ability to change the microvascular environment of regenerating nerves. One can theorize that by improving the microvascular environment-and thus improving the metabolic environment--a damaged nerve might indeed heal more effectively. Improved tissue perfusion and oxygenation during the healing and regenerating phase in neural damage could theoretically reduce such factors as synkinesis and improve the integrity of the nerve. We undertook this study to evaluate the effect of endothelial cell growth factor (ECGF), which is known to induce angiogenesis, 1 on peripheral nerve regeneration in a rat sciatic nerve model. METHODS Materials The experimental animals were 8-week-old female Sprague-Dawley rats. All test solutions consisted of ECGF (Calbiochem, San Diego, Calif.) at 3.75 mg/ml with heparin (Elkin-Sinn, Cherry Hill, NJ) at 100 pg/ml. The control solutions comprised normal saline From the Department of Otolaryngology,Bowman Gray School of Medicine, WakeForest University. Reprint requests: Dale Browne, MD, Departmentof Otolaryngology, Wake Forest University Medical Center, Medical Center Blvd., Winston-Salem,NC 27157-1034. Copyright 9 1998 by the AmericanAcademy of OtolaryngologyHead and Neck SurgeryFoundation, Inc. 0194-5998/98/$5.00 + 0 23/1/78739 178

solution with heparin at the same dilution. This protocol was approved by the Institutional Animal Care and Use Committee following guidelines of the National Society for Medical Research and was in accordance with the guidelines of the National Institutes of Health for the use of laboratory animals. Angiogenesis The ability of ECGF to induce angiogenesis was determined with the chick chorioallantoic membrane (CAM) method as described by Bo et al. e Briefly, a window was created in 3-day-old fertilized eggs (Hubbard Farms, Statesville, NC). On day 9, 50 lal of the test or control solution was placed on the CAM contained by a Silastic ring. Incubation was continued for 3 days more, after which the membrane was removed, fixed in 10% formaldehyde for 24 hours, and examined under a dissecting microscope at 25• power. The vascular density index (VDI) was determined by counting the number of vessels that crossed four concentric rings etched on a cover slip. Procedure Two groups of nine rats were used. Ketamine (80 mg/kg) combined with xylazine (12 mg/kg) was used for anesthesia. The back and leg of each animal were shaved and prepared in sterile fashion. Aseptic techniques were used throughout the operative procedure. The left sciatic nerve was exposed and freed from surrounding tissue over a length of approximately 2 cm using the posterior cutaneous thigh branch as the proximal landmark and the popliteal fossa as the distal landmark. By microsurgical technique, a 0.5 cm section of nerve was then resected, inverted, and reanastomosed as a cable graft. The nerves were approximated with 10-0

OtolaryngologyHead and Neck Surgery

SMITH and BROWNE 179

Volume 118 Number 2

Graft

Intact

Group Exp (Graft) vs. Cont (Graft) - NS

Fig. 2. Nerve latency conduction results.Graft demonstrates slightlyimproved latency in experimental group and significantdifference in latency comparing operated with nonoperated nerves.

90

9

80-

Exp

[]

Cont )

70 6O

Fig. 1. Surgical field (small arrows, anastomosis; large arrow, osmotic pump catheter). Rat sciatic nerve demonstrating anastomosis sheathed in Silastic sheath with attached osmotic p u m p catheter.

~6O>

2O 10 0.--

nylon in the epineural sheath using nerve clamps so that closure could be performed without tension. A 1.0 cm length of Silastic tube (internal diameter 0.03 in), which had been placed previously over the distal end of the nerve, was positioned over the cable-grafted segment. A 2-week delivery osmotic pump (model 2002, Alzet Corporation, Palo Alto, Calif.), which had been primed according to the recommendations of the manufacturer, was placed in a subcutaneous pocket in the back of the animal. The pump had a reservoir volume of 200 pl with a calibrated delivery rate of 0.5 pl/hr. A Silastic catheter was tunneled under the skin and secured with suture in the Silastic sheath, allowing the test or control solution to bathe the cable-grafted segment of nerve over a 2week period (Fig. 1). With aseptic technique, the pumps were removed from the subcutaneous pocket at 3 weeks, but the catheter was not removed from the nerve graft site. At 5 weeks, the animals were again anesthetized, and both the right and the left sciatic nerves were exposed for nerve conduction studies. After the studies, the animals were killed. The catheters were examined to ensure that none had migrated from the nerve; in no case did this happen. Nerve Conduction Studies

Nerve conduction latency velocities (NCL) were measured on both legs using a DISA model 1500 elec-

Graft

Intact Group Exp (Graft) vs, Cont (Graft) - *P<0.005

Fig. 3. Vascular counts. Graph demonstrates significant difference in the vascularity of grafted nerves treated with ECGF versus control and no difference in vascularity of nonoperated legs between treatment and control groups.

tromyograph (Dantec). The stimulus was consistently applied at a point just distal to the posterior cutaneous thigh branch with electrodes having been placed in the extensor muscles of the foot. Comparisons were made between the grafted and intact nerves within an animal, and between experimentally treated animals and control animals. Histologic Studies

The grafted and intact nerves were resected, including a cuff of nerve proximal and distal to the cablegrafted site. The nerves were fixed in 10% formaldehyde and embedded in paraffin. Mid-graft sections, and sections in the corresponding area of the intact nerve were obtained and stained with hematoxylin and eosin (H and E) and Verhoff silver stain. Total vascular counts within the nerve (excluding those within the epineurium on the H and E-stained sections) were performed

OtolaryngologyHead and NeckSurgery February 1998

180 SMITHand BROWNE

Fig. 4. Histologic specimen demonstrating angiogenesis. A, Experimental; B, control (arrow indicates vascular structure). Photo demonstrates significantly more vascular structures in nerve treated with ECGF. under • power. The number of regenerating myelinating axons in three different, randomly selected, high-power fields (hpf) at • oil immersion was determined on the Verhoff silver-stained sections. Axonal counts were performed on the experimental and control groups, but not on the intact nerves. Statistical Analyses Differences between the study populations were determined using the student t test and the repeated analysis of variance. All counts were performed in a blinded manner. RESULTS Angiogenesis Endothelial cell growth factor induced angiogenesis in the CAM. The experimental group (n = 5) had a VDI of 426 _+7, with the control group (n = 4) having a VDI of 242 _+ 8. This difference was statistically significant (p < 0.005). Nerve Conduction Studies The average NCL in the experimental group, grafted side, was 15.3 _+5.3 msec, whereas in the control group, grafted side, it was 17.2 _+ 3.3 msec. Although tending toward improved function, this difference proved to b e statistically insignificant. No differences were noted between the experimental and control groups in the

intact nerves, although a difference was noted when the intact and grafted nerves were compared (Fig. 2). Vascular Counts The total number of vascular structures was counted within the epineural sheath in the experimental and control groups of both the intact and the grafted nerves. The experimentally treated group had significantly more vascular structures (average of 69 _ 14 vessels/nerve) than the control grafted group (average 36 _+23 vessels/nerve) (p < 0.005). No differences were noted when comparing the vessels in the intact nerves between the groups or between intact nerves and the grafted nerves in the control group (Figs. 3 and 4). Axon Counts No differences in axon counts were noted between the control and experimental groups, with the control group having an average of 129 + 43 axons/3 hpf and the experimental group an average of 116 _+29 axons/3 hpf (Fig. 5). DISCUSSION The role of growth factors in the biochemical regulation of tissue growth and regeneration is an interesting and challenging area of study facing modem research. Many mitogenic factors have been isolated from a number of sources, including epidermal growth

OtolaryngologyHead and Neck Surgery Volume 118 Number 2

factor from a murine submandibular gland, 3 fibroblast growth factor (FGF) from a bovine pituitary gland, 4 and ECGF, used in this study, from bovine hypothalamus. 1 Topically applied epidermal growth factor has been shown to improve wound healing in skin grafts on human subjects. 5 Fibroblast growth factor improves survival of irradiated free bone grafts, 6 demonstrates mitogenic activity for aortic endothelial cells and induces angiogenesis in the CAM model, 7 and stabilizes the survival of endothelial cells in culture. 8 Endothelial cell growth factor was selected for this study because of its known ability to induce angiogenesis.1 Although distinct from FGF, ECGF also has been shown to be mitogenic for endothelial cells in vitro. Heparin was used in this study because it has been demonstrated to potentiate the endothelial mitogenic activity of ECGF by increasing the affinity of the complex for the cell surface receptor. 8 Heparin also interacts structurally with platelet-derived growth factor, 9 insulin-like growth factor I, 1~ and FGF. 11 In an excellent study by Hom and Assefa, 12 ECGF in combination with heparin improved the flap viability of rabbit ear skin flaps, presumably through improved angiogenesis. Angiography demonstrated both an increased number and in increased size of vessels in the treated group. Other growth factors have been shown to induce angiogenesis as well. Santos et al. t3 found that nerve growth factor (NGF) induced angiogenesis in sectioned nerves allowed to regenerate in silicone tube implants. Spector et a1.14 found that treatment of a severed facial nerve (in Silastic tube implants) with NGF resulted in increased myelination, increased vascularity, and a more mature fascicular pattern in the regenerated nerve. The role of growth factors in tumor angiogenesis has also been investigated. Isolates of ECGF from several human tumor cell lines were shown to be mitogenic for fetal bovine heart and aortic endothelium, thus supporting such a role. 15 The mechanism by which ECGF induced angiogenesis in this model is not known, although several possibilities exist. Schreiber et al. 8 demonstrated that EC.GF interacted directly with a specific high affinity binding site on an endothelial cell surface receptor that could be blocked with specific monoclonal antibodies to ECGF. Like FGF and transforming growth factor-alpha, ECGF may exert a direct proliferative effect on the endothelial cell to promote new vascular growth, t6 Endothelial cell growth factor also may stimulate the release of other, perhaps uncharacterized, growth factors that secondarily induce angiogenesis, or it may enhance other such growth factors delivered to the wound by inflammatory cells. 17 Finally, angiogenesis can be stimulated simply by nonspecific inflammation. However, one would not

SMITH and BROWNE 181

200-

150

~

ii !ii ii84

100-

50-

=

..,~ r

E~

Exp

Group NS

Fig. 5. Axon counts. Graph demonstrates no difference in total axon counts comparing nerves treated with ECGF and control nerves.

expect a difference between the control and the experimental groups in this study, because both were subject to the same degree of inflammatory response. The results obtained in this study support the role of ECGF on angiogenesis in a rat sciatic nerve model, clearly demonstrating an increase in vascularity in the treated group. Often in surgery, the vascular supply to a nerve is compromised, either during isolation of the nerve from surrounding tissues, or during grafting, as in this model. Therapy aimed at improving vascularity of a nerve may improve functional outcome. Although there was a tendency toward improved functional outcome as determined by nerve conduction latency, it was not significant in this study design. This may have been a reflection of the size of the study population or the result of other possible reasons discussed below. The rat sciatic nerve was selected because it has a well described vascular supply 18 and offers an easily manipulated surgical specimen and field. Our model, using the osmotic pump, was successful both in its simplicity and outcome. Although the study was controlled, there are many questions to be raised. The growth factor was delivered at the basal temperature of the animal; it is not known whether ECGF maintains activity over such a time period and temperature. Although we could identify no studies on the stability of ECGF as it relates to degradation over time and with temperatures, Williams 19 demonstrated no loss of biological activity of NGF at 37 ~ C in vitro after one month or in vivo after 2 weeks of infusion. Schoenle et al. 2~ demonstrated stability of insulin growth factor II at 6 days at 37 ~ C. A future study with ECGF might involve determining the effect of ECGF on angiogenesis using the CAM model, as described by B o e t al.,2 evaluating the effect of time and temperature on the stability of the growth factor. Also, the growth factor may only need to be present in a first-pass type effect, because in a study of FGE

OtolaryngologyHead and Neck Surgery February 1998

182 SMITHand BROWNE

Hebda et al. 21 demonstrated no increase in amplitude of effect with repeated exposure to growth factor. The duration of exposure to the growth factor, as well as the times to the killing of the animals and evaluation of nerve regeneration were 2 and 5 weeks, respectively. Additional studies might include evaluation of nerve regeneration at more prolonged periods of healing. Our results indicated a tendency toward decreased nerve conduction latency, possibly implying an improvement in functional outcome. Perhaps, with more time for myelination and fascicular reorganization, the conduction properties of the treated cable-grafted nerves might have improved. Finally, the concentration of ECGF used in this study was much higher than that reported by Maciag et al.,1 who showed the growth factor to be mitogenic for human endothelial cells at 150 pg/ml, and double that reported by Horn and Assefa, lz who showed angiogenesis in the rabbit skin flap model at 1800 ~tg/ml. Other concentrations of ECGF, in addition to other exposure times and delivery methods, may prove to be more effective in angiogenesis and neural regeneration. The use of various growth factors to promote wound healing may one day prove clinically applicable, although further studies are necessary to elucidate all mechanisms of action of the factors. We hope to continue this work by further evaluating the ability of ECGF to enhance peripheral nerve regeneration. Such studies include characterizing the role of temperature and time of delivery on the stability of ECGF, studies of other mechanisms of delivery, and studies to determine the mechanism of action of ECGF as it relates to angiogenesis.

10.

We thank Melissa Anderson and Jai Ryu, PhD, for their

16.

assistance in the performance of this study, and Joan Schnute for the histologic preparations.

REFERENCES l. Maciag T, Cerundolo J, Ilsley S, Kelley PR, Forand R. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc Natt Acad Sci USA 1979;76:5674-8. 2. Bo WJ, Mercuri M, Tucker R, Bond MG. The human carotid atherosclerotic plaque stimulates angiogenesis on the chick chorioallantoic membrane, Atherosclcrosis 1992;94:71-8. 3. Cohen S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in new-born animal. J Biol Chem 1962;237:1555-62. 4. Gospodarowicz D, Bialecki H, Greenburg G. Purification of the

5.

6.

7.

8.

9.

11.

12.

13.

14.

15.

17.

18.

19.

20.

21.

fibroblast growth factor activity from bovine brain. J Biol Chem 1978;253:3736-43. Brown GL, Nanney LB, Griffen J, et al. Enhancement of wound healing by topical treatment with epidermal growth factor. New Engl J Med 1989;321:76-9. Eppley BL, Connolly DT, Winkelmann T, Sadove AM, Heuvelman D, Feder J. Free bone graft reconstruction of irradiated facial tissue: experimental effects of basic fibroblast growth factor stimulation. Hast Reconstr Surg 1991 ;88:1-11. Thomas KA, Rios-Candelore M, Gimenez-Gallego G, et al. Pure bruin-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1. Proc Natl Acad Sci USA 1985;82:6409-13. Schreiber AB, Kenney J, Kowalski WJ, et al. Interaction of endothelial cell growth factor with heparin: characterization by receptor and antibody recognition. Proc Natl Acad Sci USA 1985;82:6138-42. Deuel TF, Huang JS, Proffitt RT, Baenziger JU, Chang D, Kennedy BB. Human platelet-derived growth factor. Purification and resolution into two active protein fractions. J Biol Chem 1981 ;256:8896-9. Clemmons DR, Underwood LE, Chatelain PG, van Wyck JJ. Liberation of immunoreactive somatomedin-C from its binding proteins by proteolytic enzymes and heparin. J Clin Endocrinol Metab 1983;56:384-9. Gospodarowicz D, Cheng J, Lui G-M, Baird A, Bohlent P. Isolation of brain fibroblast growth factor by heparin-sepharnse affinity chromatography: identity with pituitary fibroblast growth factor. Proc Natl Acad Sci USA 1984;81:6963-7. Hom DB, Assefa G. Effects of endothelial cell growth factor on vascular compromised skin flaps. Arch Otolaryngol Head Neck Surg 1992;118:624-8. Santos PM, Winterowd JG, Allen GG, Bothwell MA, Rubel EW. Nerve growth factor: increased angiogenesis without improved nerve regeneration. Otolaryngol Head Neck Surg 1991;105:1225. SpectorJG, Lee P, Derby A, Frierdich GE, Neises G, Roufa DG. Rabbit facial nerve regeneration in NGF-containing silastic tubes. Laryngoscope 1993;103:548-58. 0lander JV, Marasa JC, Kimes RC, Johnston GM, Feder J. An assay measuring the stimulation of several types of bovine endothelial cells by growth factor(s) derived from cultured human tumor cells. In Vitro Cell Dev Biol Anim 1982;18(2):99107. Folkman J, Klagsbrun J. Angiogenic factors. Science 1987;235: 442-7. Madtes DK, Raines EW, Sakariassen KS, et al. Induction of transforming growth factor-~ in activated human alveolar macrophages. Cell 1988;53:285-93. Bell MA, Weddell GM. A descriptive study of the blood vessels of the sciatic nerve in the rat, man and other mammals. Brain 1984;107:871-98. Williams LR. Exogenous nerve growth factor stimulates choline acetyltransferase activity in aging Fischer 344 male rats. Neurobiol Aging 1991;12:39-46. Schoenle E, Zapt J, Haari C, Steiner T, Froesch ER. Comparison of in vivo effects of insulin-like growth factors I and II and of growth hormone in hypophysectomized rats. Acta Endocrinologica 1985;108:167-74. Hebda PA, Klingbeil CK, Abraham JA, Fiddes JC. Basic flbroblast growth factor stimulation of epidermal wound healing in pigs. J Invest Dermatol 1990;95:626-31.