Amniotic membrane can act as a biological bandage contact lens to the ocular surface, with therapeutic anti-inflammatory properties. The analgesic benefits of this intervention are believed to be due to mechanical protection. Amniotic membrane is also used in the treatment of neurotrophic corneal disease. Substance P is a neurotransmitter released by C-terminal nerve endings. It mediates acute inflammation and is known to play a role in modulating pain sensation. Substance P is also known to promote proliferation of various cell types, including epithelial and nerve progenitor cells.1,2 In this in vitro pilot study, we investigated the relationship between amniotic membrane and substance P.
This study was approved to use surplus tissue from 6 previously harvested amniotic membranes. The original samples were washed, prepared, and stored using standard techniques to ensure no contamination from blood. Residual pieces were defrosted and washed 4 times in phosphate-buffered saline. Sections measuring 0.5 × 0.25 cm were used throughout. The tissue was sonicated for 1 minute in phosphate-buffered saline and 0.5% Triton X and spun in a Jouan microfuge for 10 minutes at 400 rpm. This was analyzed using a Parameter Substance P competitive enzyme immunoassay (R&D Systems).
Substance P was present in amniotic membrane samples at a range between 4000 and 6000 pg/mL. (Samples were diluted 1:50 to enable analysis.) In comparison, substance P levels in independent saliva and serum samples from volunteers in the research team ranged between 8 and 200 pg/mL in saliva and between 1200 and 1300 pg/mL in serum. We were unable to demonstrate uptake of substance P following incubation with the saliva or serum by this method owing to the very high concentration of substance P in amniotic membrane.
This pilot study demonstrates that substance P is present in very high concentrations in amniotic membrane. This exceeded the concentrations in our control sources of serum or saliva and was found despite several months of storage. Substance P has been identified in amniotic fluid in mid to late gestation.3 Further investigation would be required to elucidate whether this is the source of amniotic membrane's substance P. Urine is known to be a rich source of substance P, and fetal urine is produced from 12 weeks after conception. We wonder whether protective absorption of substance P by amniotic membrane from amniotic fluid in utero is the source of our findings.
Eyedrops containing substance P and insulinlike growth factor have been tried with some success in therapy for nonhealing neurotrophic corneal ulcers. Substance P and insulinlike growth factor are believed to act synergistically in promoting corneal epithelial proliferation.4,5 Our positive identification of substance P in amniotic membrane may offer further insights into its mode of action in healing the ocular surface. Tseng et al6 investigated 31 eyes of 26 patients with cytologically proven limbal deficiency who received amniotic membrane transplants, and all showed rapid epithelialization and reduced inflammation except for the 2 eyes with atopy. It has been our clinical observation that some atopic patients can experience unexplained ocular surface irritation following treatment with amniotic membrane. Substance P is known to degranulate mast cells.1 We wonder whether substance P released by amniotic membrane could provide a partial explanation for this phenomenon in atopic individuals and could have contributed to the failure of treatment in the 2 patients of Tseng and colleagues.
We have demonstrated that substance P is present in very high concentrations in amniotic membrane. Further study to identify the hitherto unrealized substances involved in the scavenging of damaging agents by amniotic membrane may permit development of specific topical preparations to treat the ocular surface.
Correspondence: Dr Lockington, Tennent Institute of Ophthalmology, Gartnavel General Hospital, 1053 Great Western Rd, Glasgow G12 0YN, Scotland (davidlockington@hotmail.com).
Financial Disclosure: None reported.
1.Paus R, Heinzelmann T, Robicsek S, Czarnetzki BM, Maurer M. Substance P stimulates murine epidermal keratinocyte proliferation and dermal mast cell degranulation in situ.
Arch Dermatol Res. 1995;287(5):500-5027542862
PubMedGoogle ScholarCrossref 2.Park SW, Yan YP, Satriotomo I, Vemuganti R, Dempsey RJ. Substance P is a promoter of adult neural progenitor cell proliferation under normal and ischemic conditions.
J Neurosurg. 2007;107(3):593-59917886560
PubMedGoogle ScholarCrossref 3.Sanfilippo JS, Botti JJ, Wild RA, Osuamkpe CO. Amniotic fluid levels of substance P.
J Reprod Med. 1992;37(8):733-7361279166
PubMedGoogle Scholar 4.Brown SM, Lamberts DW, Reid TW, Nishida T, Murphy CJ. Neurotrophic and anhidrotic keratopathy treated with substance P and insulinlike growth factor 1.
Arch Ophthalmol. 1997;115(7):926-9279230840
PubMedGoogle ScholarCrossref 5.Yamada N, Matsuda R, Morishige N,
et al. Open clinical study of eye-drops containing tetrapeptides derived from substance P and insulin-like growth factor-1 for treatment of persistent corneal epithelial defects associated with neurotrophic keratopathy.
Br J Ophthalmol. 2008;92(7):896-90018511539
PubMedGoogle ScholarCrossref 6.Tseng SC, Prabhasawat P, Barton K, Gray T, Meller D. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency.
Arch Ophthalmol. 1998;116(4):431-4419565039
PubMedGoogle Scholar